Glucoamylase variants

ABSTRACT

The invention relates to a variant of a parent fungal glucoamylase, which exhibits improved thermal stability and/or increased specific activity using saccharide substrates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/524,693filed on Sep. 21, 2006, now U.S. Pat. No. 7,833,772, which is acontinuation of U.S. application Ser. No. 10/038,723 filed on Jan. 2,2002, now U.S. Pat. No. 7,122,365, which is a divisional of U.S.application Ser. No. 09/351,814 filed on Jul. 12, 1999, now U.S. Pat.No. 6,352,851, which claims priority under 35 U.S.C. 119 of Danishapplication nos. PA 1998 00937 and PA 1998 01667 filed on Jul. 15, 1998and Dec. 17, 1998, respectively, and U.S. provisional application Nos.60/093,528 and 60/115,545 filed on Jul. 21, 1998 and Jan. 12, 1999,respectively, the contents of which are fully incorporated herein byreference.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel glucoamylase variants (mutants)of parent AMG, in particular with improved thermal stability and/orincreased specific activity suitable for, e.g., starch conversion, e.g.,for producing glucose from starch. More specifically, the presentinvention relates to glucoamylase enzyme variants and the use of suchvariant enzymes.

BACKGROUND OF THE INVENTION

Glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) is an enzymewhich catalyzes the release of D-glucose from the non-reducing ends ofstarch or related oligo- and polysaccharide molecules. Glucoamylases areproduced by several filamentous fungi and yeasts, with those fromAspergillus being commercially most important.

Commercially, glucoamylase is used to convert corn starch which isalready partially hydrolyzed by an alpha-amylase to glucose. The glucoseis further converted by glucose isomerase to a mixture composed almostequally of glucose and fructose. This mixture, or the mixture furtherenriched with fructose, is the commonly used high fructose corn syrupcommercialized throughout the world. This syrup is the world's largesttonnage product produced by an enzymatic process. The three enzymesinvolved in the conversion of starch to fructose are among the mostimportant industrial enzymes produced.

One of the main problems that exist with regard to the commercial use ofglucoamylase in the production of high fructose corn syrup is therelatively low thermal stability of glucoamylase. Glucoamylase is not asthermally stable as alpha-amylase or glucose isomerase and it is mostactive and stable at lower pH's than either alpha-amylase or glucoseisomerase. Accordingly, it must be used in a separate vessel at a lowertemperature and pH.

Glucoamylase from Aspergillus niger has a catalytic (aa 1-440) and astarch binding domain (aa 509-616) separated by a long and highlyO-glycosylated linker (Svensson et al., 1983, Carlsberg Res. Commun. 48:529-544 and Svensson et al., 1986, Eur. J. Biochem. 154: 497-502). Thecatalytic domain (aa 1-471) of glucoamylase from A. awamori var. X100adopt an (α/α)₆-fold in which six conserved α→α loop segments connectthe outer and inner barrels (Aleshin et al., 1992, J. Biol. Chem. 267:19291-19298). Crystal structures of glucoamylase in complex with1-deoxynojirimycin (Harris et al., 1993, Biochemistry 32: 1618-1626) andthe pseudotetrasaccharide inhibitors acarbose andD-gluco-dihydroacarbose (Aleshin et al., 1996, Biochemistry 35:8319-8328) furthermore are compatible with glutamic acids 179 and 400acting as general acid and base, respectively. The crucial role of theseresidues during catalysis has also been studied using proteinengineering (Sierks et al., 1990, Protein Engng. 3: 193-198; Frandsen etal., 1994, Biochemistry 33: 13808-13816). Glucoamylase-carbohydrateinteractions at four glycosyl residue binding subsites, −1, +1, +2, and+3 are highlighted in glucoamylase-complex structures (Aleshin et al.,1996, Biochemistry 35: 8319-8328) and residues important for binding andcatalysis have been extensively investigated using site-directed mutantscoupled with kinetic analysis (Sierks et al., 1989, Protein Engng. 2:621-625; Sierks et al., 1990, Protein Engng. 3: 193-198; Berland et al.,1995, Biochemistry 34: 10153-10161; Frandsen et al., 1995, Biochemistry34: 10162-10169.

Different substitutions in A. niger glucoamylase to enhance the thermalstability have been described: i) substitution of alpha-helicalglycines: G137A and G139A (Chen et al., 1996, Prot. Engng. 9: 499-505);ii) elimination of the fragile Asp-X peptide bonds, D257E and D293E/Q(Chen et al., 1995, Prot. Engng. 8: 575-582); prevention of deamidationin N182 (Chen et al., 1994, Biochem. J. 301: 275-281); iv) engineeringof additional disulphide bond, A246C (Fierobe et al., 1996, Biochemistry35: 8698-8704; and v) introduction of Pro residues in position A435 andS436 (Li et al., 1997, Protein Engng. 10: 1199-1204. Furthermore ClarkFord presented a paper on Oct. 17, 1997, ENZYME ENGINEERING 14,Beijing/China Oct. 12-17, 1997, Abstract number: Abstract book p. 0-61.The abstract suggests mutations in positions G137A, N20C/A27C, and S30Pin a (not disclosed) Aspergillus awamori glucoamylase to improve thethermal stability.

Additional information concerning glucoamylase can be found on anInternet homepage (public.iastate.edu/˜pedro/glase/glase.html)“Glucoamylase WWW page” (Last changed 1997 Oct. 8) by Pedro M. Coutinhodiscloses informations concerning glucoamylases, including glucoamylasesderivable from Aspergillus strains. Chemical and site-directedmodifications in the Aspergillus niger glucoamylase are listed.

BRIEF DISCLOSURE OF THE INVENTION

The object of the present invention is to provide improved glucoamylasevariants with improved thermostability and/or increased specificactivity suitable for use in, e.g., the saccharification step in starchconversion processes.

The term “a glucoamylase variant with improved thermostability” means inthe context of the present invention a glucoamylase variant which has ahigher T_(1/2) (half-time) than the corresponding parent glucoamylase.The determination of T½ (Method I and Method II) is described below inthe “Materials & Methods” section.

The term “a glucoamylase variant with increased specific activity” meansin the context of the present invention a glucoamylase variant withincreased specific activity towards the alpha-1,4 linkages in thesaccharide in question. The specific activity is determined as k_(cat)or AGU/mg (measured as described below in the “Materials & Methods”section). An increased specific activity means that the k_(cat) orAGU/mg values are higher when compared to the k_(cat) or AGU/mg values,respectively, of the corresponding parent glucoamylase.

The inventors of the present invention have provided a number ofimproved variants of a parent glucoamylase with improved thermostabilityand/or increased specific activity in comparison to the parentcorresponding enzyme. The improved thermal stability is obtained bysubstituting selected positions in a parent glucoamylase. This will bedescribed in details below.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used. For ease ofreference, glucoamylase variants of the invention are described by useof the following nomenclature:

-   -   Original amino acid(s):position(s):substituted amino acid(s)

According to this nomenclature, for instance the substitution of alaninefor asparagine in position 30 is shown as:

-   -   Ala30Asn or A30N        a deletion of alanine in the same position is shown as:    -   Ala30* or A30*        and an insertion of an additional amino acid residue, such as        lysine, is shown as:    -   Ala30AlaLys or A30AK

A deletion of a consecutive stretch of amino acid residues, such asamino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

Where a specific glucoamylase contains a “deletion” in comparison withother glucoamylases and an insertion is made in such a position this isindicated as:

-   -   *36Asp or *36D        for an insertion of an aspartic acid in position 36.

Multiple mutations are separated by plus signs, i.e.:

-   -   Ala30Asp+Glu34Ser or A30N+E34S        representing mutations in positions 30 and 34 substituting        alanine and glutamic acid for asparagine and serine,        respectively. Multiple mutation may also be separated as        follows, i.e., meaning the same as the plus sign:    -   Ala30Asp/Glu34Ser or A30N/E34S

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as

-   -   A30N,E or A30N/E, or A30N or A30E

Furthermore, when a position suitable for modification is identifiedherein without any specific modification being suggested, it is to beunderstood that any amino acid residue may be substituted for the aminoacid residue present in the position. Thus, for instance, when amodification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of: R, N, D, A, C,Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the plasmid pCAMG91 containing the Aspergillus niger G1glucoamylase gene.

DETAILED DISCLOSURE OF THE INVENTION

A goal of the work underlying the present invention was to improve thethermal stability and/or increase the specific activity of particularglucoamylases which are obtainable from fungal organisms, in particularstrains of the Aspergillus genus and which themselves had been selectedon the basis of their suitable properties in starch conversion oralcohol fermentation.

Identifying Positions and/or Regions to be Mutated to Obtain ImprovedThermostability and/or Increased Specific Activity

Molecular dynamics (MD) simulations indicate the mobility of the aminoacids in the protein structure (see McCammon and Harvey, 1987, “Dynamicsof proteins and nucleic acids”. Cambridge University Press). Suchprotein dynamics are often compared to the crystallographic B-factors(see Stout, G H and Jensen, L H, 1989, “X-ray structure determination”,Wiley). By running the MD simulation at different protonation states ofthe titrate able residues, the pH related mobility of residues aresimulated. Regions having the highest mobility or flexibility (hereisotropic fluctuations) are selected for random mutagenesis. It is hereunderstood that the high mobility found in certain areas of the protein,can be thermally improved by substituting residues in these residues.The substitutions are directed against residues that will change thedynamic behaviour of the residues to e.g., bigger side-chains and/orresidues which have capability of forming improved contacts to residuesin the near environment. The AMG from Aspergillus niger was used for theMD simulation. How to carry out MD simulation is described in theMaterials & Methods” section below.)

Regions found by Molecular dynamics (MD) simulations to be suitable formutation when wanting to obtain improved thermal stability and/orincreased specific activity are the following:

Region: 1-18,

Region: 19-35,

Region: 73-80,

Region: 200-212,

Region: 234-246,

Region: 334-341,

Region: 353-374,

Region: 407-411,

Region: 445-470,

Regions found to be of interest for increasing the specific activityand/or improved thermostability are the regions in proximity to theactive site. Regions positioned in between the alpha-helixes, and whichmay include positions on each side of the N- and C-terminal of thealpha-helixes, at the substrate binding site is of importance for theactivity of the enzyme. These regions constitute the following regions:

Region: 40-62,

Region: 93-127,

Region: 170-184,

Region: 234-246,

Region: 287-319,

Region: 388-414.

Rhizopus, Talaromyces, such as Talaromyces emersonii (disclosed in WO99/28448), and Thielavia have high specific activity towardsmaltodextrins, including maltose and maltohepatose. Therefore, regionsbeing of special interest regarding (transferring) increased specificactivity are:

Region: 200-212,

Region: 287-300,

Region: 305-319.

The present inventors have found that it is in fact possible to improvethe thermal stability and/or to increase the specific activity of aparent glucoamylase by modification of one or more amino acid residuesof the amino acid sequence of the parent glucoamylase. The presentinvention is based on this finding.

Accordingly, in a first aspect the present invention relates to animproved variant of a parent glucoamylase comprising one or moremutations in the regions and positions described further below.

Parent Glucoamylases

Parent glucoamylase contemplated according to the present inventioninclude fungal glucoamylases, in particular fungal glucoamylasesobtainable from an Aspergillus strain, such as an Aspergillus niger orAspergillus awamori glucoamylases and variants or mutants thereof,homologous glucoamylases, and further glucoamylases being structurallyand/or functionally similar to SEQ ID NO: 2. Specifically contemplatedare the Aspergillus niger glucoamylases G1 and G2 disclosed in Boel etal., 1984, “Glucoamylases G1 and G2 from Aspergillus niger aresynthesized from two different but closely related mRNAs”, EMBO J. 3(5):1097-1102. The G2 glucoamylase is disclosed in SEQ ID NO: 2. The G1glucoamylase is disclosed in SEQ ID NO: 13. Another AMG backbonecontemplated is Talaromyces emersonii, especially Talaromyces emersoniiDSM disclosed in WO 99/28448 (Novo Nordisk).

Commercially Available Parent Glucoamylases

Commercially available parent glucoamylases include AMG from NovoNordisk, and also glucoamylase from the companies Genencor, Inc. USA,and Gist-Brocades, Delft, The Netherlands.

Glucoamylase Variants

In the first aspect the invention relates to a variant of a parentglucoamylase comprising one or more mutation(s) in the followingpositions(s) or region(s) in the amino acid sequence shown in SEQ ID NO:2:

Region: 1-18,

Region: 19-35,

Region: 40-62,

Region: 73-80,

Region: 93-127,

Region: 170-184,

Region: 200-212,

Region: 234-246,

Region: 287-319,

Region: 334-341,

Region: 353-374,

Region: 388-414,

Region: 445-470,

and/or in a corresponding position or region in a homologousglucoamylase which displays at least 60% homology with the amino acidsequences shown in SEQ ID NO: 2, with the exception of the followingsubstitutions: N20C, A27C, S30P, Y48W, Y50F, W52F, R54K/L, D55G/V, G57A,K108R, D112Y, Y116A/W, S119C/W/E/G/Y/P, W120H/L/F/Y, G121T/A, R122Y,P123G, Q124H, R125K, W170F, N171S, Q172N, T173G, G174C, Y175F, D176N/E,L177H/D, W178R/D, E179Q/D, E180D/Q, V181D/A/T, N182A/D/Q/Y/S, G183K,S184H, W212F, R241K, A246C, D293E/Q, A302V, R305K, Y306F, D309N/E,Y312W, W317F, E389D/Q, H391W, A392D, A393P, N395Q, G396S, E400Q/C,Q401E, G407D, E408P, L410F, S411A/G/C/H/D, S460P

In an embodiment the region mutated is the Region: 1-18.

Specific preferred positions contemplated include one or more of:

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18.

Specific mutations include one or more of: A1V, T2P/Q/R/H/M/E/K, L3N,N9A, A11E/P, I18V.

Preferred combinations of mutations include one or more of:

A1V+L66R+Y402F+N427S+S486G,

T2K+S30P+N427M+S44G+V470M,

T2E+T379A+S386K+A393R,

T2Q-A11P+S394R,

T2R+L66V+S394P+Y402F

T2M+N9A+T390R+D406N+L410R,

T2R+S386R+A393R,

A11P+T2Q-S394R,

A11E+E408R,

I18V+T51S+S56A+V59T+L60A.

In an embodiment the region mutated is the Region: 19-35.

Preferred sub-regions include one or more of: 21-26, 31-35.

Specific preferred positions contemplated include one or more of:

19, 21, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35.

Specific mutations include one or more of: L19N, N20T, G23A, A24S/T,D25S/T/R, G26A, A27S/T, W28R/Y, S30T/N, G31A, A32V, D33R/K/H, S34N.

In an embodiment the region mutated is the Region: 40-62.

Preferred sub-regions include one or more of: 40-47, 58-62.

Specific preferred positions contemplated include one or more of:

40, 41, 42, 43, 44, 45, 46, 47, 49, 51, 53, 56, 58, 59, 60, 61, 62.

Specific mutations include one or more of:

S40C/A/G,

T43R,

T51S/D,

T53D,

S56A/C,

V59T/A,

L60A.

Preferred combinations of mutations include one or more of:

T51S+S56A+V59T+L60A+I18V

V59A+A393R+T490A+PLASD(N-terminal extension).

In an embodiment the region mutated is the Region: 73-80.

Specific preferred positions contemplated include one or more of:

73, 74, 75, 76, 77, 78, 79, 80.

Specific mutations include one or more of:

S73P/D/T/N/Q/E,

L74I/D,

L75A/R/N/D/C/Q/E/G/H/I/K/M/P/S/T/V, preferred are I/N/D,

S76A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred are T/A,

T77V/T,

I78V,

E79A/R/N/D/C/Q/G/H/I/L/K/F/M/P/S/T/Y/V, preferred are Q/R/K,

N80A/R/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred are H/D/E/R/K/T/S/Y.

In an embodiment the region mutated is the Region: 93-127.

In an additional embodiment the sub-region is: Region: 93-124.

Preferred sub-regions include one or more of: 93-107, 109-111, 113-115.

Specific preferred positions contemplated include one or more of:

93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 109, 110,111, 113, 114, 115, 117, 118, 126, 127.

Specific mutations include one or more of:

N93T,

P94V,

S95N,

D97S,

L98S/P,

S100T/D,

A102S/*,

P107M/L/A/G/S/T/V/I,

N110T,

V111P,

D112N,

E113M/A,

T114S,

A115Q/A,

Y116F,

S119A/R/N/D/Q/H/I/L/K/F/M/T/V, preferred is A,

R122A/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/V,

G127A.

Preferred combinations of mutations include one or more of:

S119P+G447S,

S119P+Y402F,

S119P+A393R,

S119P+I189T+223F+F227Y+Y402F

S119P+T416H+Y402F+Y312Q.

In an embodiment the region mutated is the Region: 170-184.

Specific mutations include one or more of:

N171R/K/Q/E/W/F/Y,

Q172A/R/N/D/C/E/G/H/I/L/K/F/M/P/S/T/W/Y/V,

T173K/R/S/N/Q,

G174A,S,

Y175N/Q/D/E,

D176L,

L177I,

E180N/M,

V181I/T,

N182R/C/E/G/H/I/L/K/M/P/T/W/Y/V,

G183A,

S184D/N/E/Q/L/I/T/R/K.

In an embodiment the region mutated is the Region: 200-212.

Preferred sub-regions include: 200-211.

Specific preferred positions contemplated include one or more of:

200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211.

Specific mutations include one or more of: A201D, F202L, A203L, T204K,A205R/S, V206L/N, G207N, S208H/T/D, S209T, S211P, W212N/A/T.

In an embodiment the region mutated is the Region: 234-246.

Preferred sub-regions include one or more of: 234-240, 242-245.

Specific preferred positions contemplated include one or more of:

234, 235, 236, 237, 238, 239, 240, 242, 243, 244, 245.

Specific mutations include one or more of:

L234A/R/N/D/C/Q/E/G/H/I/K/M/F/P/S/T/W/Y/V.

A235S,

F237Y/H/N/D,

D238T/S,

S239A/R/N/D/C/G/H/I/L/F/P/S/T/Y/V,

S240G,

S242S/P/T/A/Y/H/N/D,

G243S/P/T/A/Y/H/N/D,

K244R,

A246T.

Preferred combinations of mutations include one or more of: A246T+T721.

In an embodiment the region mutated is the Region: 287-319.

Preferred sub-regions include one or more of: 287-292, 294-301, 313-316.

Specific preferred positions contemplated include one or more of:

287, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300, 301,303, 307, 308, 310, 311, 313, 314, 315, 316, 318, 319.

Specific mutations include one or more of:

S287A/R/N/D/C/Q/E/G/H/I/L/K/M/T/V,

I288L/N/Q,

Y289F,

T290A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/V,

L291I/D/N,

N292D,

D293A/R/N/C/Q/E/G/H/I/L/K/M/S/T/V,

G294A/R/N/D/C/Q/E/H/I/L/K/M/P/S/T/V,

L295A/R/N/D/C/Q/E/G/H/K/M/S/T/V,

S296A/R/N/D/C/Q/E/G/H/I/L/K/M/T/V,

D297A/R/N/C/Q/E/G/H/I/L/K/M/P/S/T/V,

S298A/R/N/D/C/Q/E/G/H/I/L/K/F/M/T/V,

E299A/R/N/D/C/Q/G/H/I/L/K/M/S/T/V,

V301T/I,

A302R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V/, preferred S,

V303T/I,

G304A,

R305A/N/D/C/Q/E/G/H/I/L/F/M/P/S/T/W/Y/V,

Y306A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/T/W/V.

E308A/R/N/D/C/Q/G/H/I/L/K/M/P/S/T/V, preferred Q,

D309L,

T310V/S,

Y311N,

Y312Q/N,

N313T/S/G,

N315Q/E/R,

F318A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/T/W/Y/V, preferred is Y.

Preferred combinations of mutations include one or more of:

Y312Q+S119P+T416H+Y402F,

Y312Q+S119P+Y402F+T416H+S411V,

Y312Q+T416H,

N313G+F318Y.

In an embodiment the region mutated is the Region: 334-341.

Specific preferred positions contemplated include one or more of:

334, 335, 336, 337, 338, 339, 340, 341.

Specific mutations include one or more of:

D336A/R/N/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V,

K337A/R/N/D/C/Q/E/G/H/I/L/M/F/P/S/T/W/Y/V,

Q338A/R/N/D/C/G/H/I/L/F/P/S/T/Y/V,

G339S/P/T/A,

S340I/T/N/V/A/D/G,

L341F/L/I/V.

Preferred combinations of mutations include one or more of:

S340G+D357S+T360V+S386P.

In an embodiment the region mutated is the Region: 353-374.

Specific preferred positions contemplated include one or more of:

353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374.

Specific mutations include one or more of:

A353D/S,

S356P/N/D,

D357S,

A359S,

T360V,

G361S/P/T/A,

T362R,

S364A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

S365A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

S366T,

S368P/T/A,

T369A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

S371Y/H/N/D,

S372F/Y/C/L/P/H/R/I/T/N/S/V/A/D/G.

Preferred combinations of mutations include one or more of:

S356P+S366T,

D357S+T360V+S371H.

D357S+T360V+S386P+S340G.

In an embodiment the region mutated is the Region: 388-414.

Preferred sub-regions include one or more of: 397-399, 402-406, 412-414.

Specific preferred positions contemplated include one or more of:

388, 389, 390, 394, 397, 398, 399, 402, 403, 404, 405, 406, 409, 412,413, 414.

Specific mutations include one or more of:

T390R,

A393R,

S394P/R,

M398L,

S399A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/T/W/Y/V, preferred are T/Q/C,

Y402A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/V, preferred is F,

D403S,

S405T,

D406N,

E408C/R,

A409R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred is P,

L410R/I,

S411R/N/Q/E/I/L/K/F/M/P/T/W/Y/V, preferred is V,

A412C,

R413A/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V.

D414A.

Preferred combinations of mutations include one or more of:

A393R+T490A+V59A+PLASD(N-terminal extension)

S394R+T2Q+A11P,

Y402F+S411V,

Y402F+S411V+S119P,

Y402F+S411V+S119P+A393R,

Y402F+Y312Q+S119P+T416H,

E408R+S386N,

E408R+A425T+S465P+T494A,

L410R+A393R,

Y402F+Y312Q+S119P+T416H+S411V+A393R.

In an embodiment the region mutated is the Region: 445-470.

Preferred sub-regions include one or more of: 445-459, 461-470.

Specific preferred positions contemplated include one or more of:

445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470.

Specific mutations include one or more of: G447S, G456C/P, S465P.

Specific variants include variants having one or more of the followingsubstitutions: A1V, T2E/P/Q/R/H/M, L3P/N, N9A, A11P/E, I18V, L19N, N20T,G23A, A24S/T, D25S/T/R, G26A, A27S/T, W28R/Y, S30T/N, G31A, A32V,D33R/K/H, S34N, S40C, T43R, T51D/S, T53D, S56A/C, V59T/A, L60A, N93T,P94V, S95N, D97S, L98P/S, S100T/D, A102S/*, N110T, V111P, D112N,E113M/A, T114S, A115Q/G, Y116F, S119A, G127A, N182E, A201D, F202L,A203L, T204K, A205R/S, V206L/N, G207N, S208H/T/D, S209T, S211P,W212N/A/T, A246T Y312Q, N313T/S/G, A353D/S, S356P/N/D, D357S, A359S,T360V, G361S/P/T/A, T362R, S364A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,S365A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V, S366T, S368P/T/A,T369A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V, S371Y/H/N/D,S372F/Y/C/L/P/H/R/I/T/N/S/V/A/D/G, T390R, A393R, S394R/P, M398L,S399C/Q/T, Y402F, D403S, S405T, D406N, E408C/R, L410I/R, S411V, A412C,D414A, G447S, S465P.

Improved Thermal Stability

In a second aspect the invention relates to a variant of a parentglucoamylase with improved thermal stability, in particular in the rangefrom 40-80° C., preferably 60-80° C., and preferably at pH 4-5, saidvariant comprising one or more mutation(s) in the following position(s)or region(s) in the amino acid sequence shown in SEQ ID NO: 2:

Region: 1-18,

Region: 19-35,

Region: 73-80,

Region: 93-127,

Region: 170-184,

Region: 200-212,

Region: 234-246,

Region: 287-319,

Region: 334-341,

Region: 353-374,

Region: 388-414,

Region: 445-470,

and/or in a corresponding position or region in a homologousglucoamylase which displays at least 60% homology with the amino acidsequences shown in SEQ ID NO: 2, with the exception of the followingsubstitutions: N20C, A27C, S30P, A246C.

As substrate binding may improve the stability region 93-127, Region:170-184, Region: 305-319 are also contemplated for thermostabilizationaccording to the present invention.

In an embodiment the region mutated is the Region: 1-18.

Specific preferred positions contemplated include one or more of:

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18.

Specific mutations include one or more of: A1V, T2P/Q/R/H/M/E, N9A,A11E/P.

Preferred combinations of mutations include one or more of:

A1V+L66R+Y402F+N427S+S486G,

T2K+S30P+N427M+S44G+V470M,

T2E+T379A+S386K+A393R,

T2R+S386R+A393R,

A11P+T2Q-S394R,

T2Q-A11P+S394R,

T2R+L66V+S394P+Y402F,

T2M+N9A+T390R+D406N+L410R,

T2R+S386R+A393R,

A11E+E408R.

In an embodiment the region mutated is the Region: 19-35.

Preferred sub-regions include one or more of: 21-26, 31-35.

Specific preferred positions contemplated include one or more of:

19, 21, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35.

Specific mutations include one or more of: L19N, N20T, G23A, A24S/T,D25S/T/R, G26A, A27S/T, W28R/Y, S30T/N, G31A, A32V, D33R/K/H, S34N.

In an embodiment the region mutated is the Region: 73-80.

Specific preferred positions contemplated include one or more of:

73, 74, 75, 76, 77, 78, 79, 80.

Specific mutations include one or more of:

S73P/D/T/N/Q/E,

L74I/D,

L75A/R/N/D/C/Q/E/G/H/I/K/M/P/S/T/V, preferred are I/N/D,

S76A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred T/A),

T77V/T,

I78V,

E79A/R/N/D/C/Q/G/H/I/L/K/F/M/P/S/T/Y/V, preferred are Q/R/K),

N80A/R/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred are H/D/E/R/K/T/S/Y.

In an embodiment the region mutated is the Region: 93-127.

In an additional embodiment the sub-region is: Region: 93-124

Preferred sub-regions include one or more of: 93-107, 109-111, 113-115.

Specific preferred positions contemplated include one or more of:

93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 109, 110,111, 113, 114, 115, 117, 118, 126, 127.

Preferred mutations include one or more of:

P107M/L/A/G/S/T/V/I,

S119A/R/N/D/Q/H/I/L/K/F/M/T/V, preferred is A,

R122A/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/V,

Preferred combinations of mutations include one or more of:

S119P+G447S,

S119P+A393R.

In an embodiment the region mutated is the Region: 170-184.

Specific mutations include one or more of:

N171R/K/Q/E/W/F/Y,

Q172A/R/N/D/C/E/G/H/I/L/K/F/M/P/S/T/W/Y/V,

T173K/R/S/N/Q,

G174A,S,

Y175N/Q/D/E,

D176L,

L177I,

E180N/M,

V181I/T,

N182R/C/E/G/H/I/L/K/M/P/T/W/Y/V.

G183A,

S184 D/N/E/Q/L/I/T/R/K.

In an embodiment the region mutated is the Region: 200-212.

Preferred sub-regions include: 200-211.

Specific preferred positions contemplated include one or more of:

200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211.

Specific mutations include one or more of: A203L, S211P.

In an embodiment the region mutated is the Region: 234-246.

Preferred sub-regions include one or more of: 234-240, 242-245.

Specific preferred positions contemplated include one or more of:

234, 235, 236, 237, 238, 239, 240, 242, 243, 244, 245.

Specific mutations include one or more of:

L234A/R/N/D/C/Q/E/G/H/I/K/M/F/P/S/T/W/Y/V,

A235S,

F237Y/H/N/D,

D238T/S,

S239A/R/N/D/C/G/H/I/L/F/P/S/T/Y/V,

S240G,

S242S/P/T/A/Y/H/N/D,

G243S/P/T/A/Y/H/N/D,

K244R,

A246T.

Preferred combinations of mutations include one or more of: A246T+T72I.

In an embodiment the region mutated is the Region: 287-319.

Preferred sub-regions include one or more of: 287-292, 294-301, 313-316.

In an additional embodiment the sub-region include: 305-319

Specific preferred positions contemplated include one or more of:

287, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300, 301,302, 303, 307, 308, 310, 311, 313, 314, 315, 316, 318, 319.

Specific mutations include one or more of:

Y312Q,

F318A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/T/W/Y/V, preferred is Y.

Preferred combinations of mutations include one or more of:

N313G+F318Y,

Y302Q+S119P+T416H+Y402F.

In an embodiment the region mutated is the Region: 334-341.

Specific preferred positions contemplated include one or more of:

334, 335, 336, 337, 338, 339, 340, 341.

Specific mutations include one or more of:

D336A/R/N/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V.

K337A/R/N/D/C/Q/E/G/H/I/L/M/F/P/S/T/W/Y/V.

Q338A/R/N/D/C/G/H/I/L/F/P/S/T/Y/V.

G339S/P/T/A.

S340I/T/N/V/A/D/G.

L341F/L/I/V.

Preferred combinations of mutations include one or more of:

S340G+D357S+T360V+S386P.

In an embodiment the region mutated is the Region: 353-374.

Specific preferred positions contemplated include one or more of:

353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374.

Specific mutations include one or more of:

A353D/S,

S356P/N/D,

D357S,

A359S,

T360V,

G361S/P/T/A,

T362R,

S364A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

S365A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

S366T,

S368P/T/A,

T369A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

S371Y/H/N/D,

S372F/Y/C/L/P/H/R/I/T/N/S/V/A/D/G.

Preferred combinations of mutations include one or more of:

S356P+S366T,

D357S+T360V+S371H

D357S+T360V+S386P+S340G.

In an embodiment the region mutated is the Region: 388-414.

Preferred sub-regions include one or more of: 397-399, 402-406, 412-414.

In an additional embodiment the sub-region is: 407-411

Specific preferred positions contemplated include one or more of:

388, 389, 390, 394, 397, 398, 399, 402, 403, 404, 405, 406, 409, 412,413, 414.

Specific mutations include one or more of:

T390R,

A393R,

S394P/R,

S399A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/T/W/Y/V, preferred are T/Q/C,

Y402A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/V, preferred is F,

D403S,

S405T,

D406N,

E408C/R,

Q409A/R/N/D/C/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred is P,

L410I/R,

S411V,

A412C,

R413A/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V,

D414A.

Preferred combinations of mutations include one or more of:

A393R+T2R+S386R,

A393R+T490A+V59A+PLASD(N-terminal extension),

S394R+T2Q-A11P,

Y402F+T2R+L66V+S394P,

Y402F+S411V+S119P,

Y402F+S411V,

Y402F+312Q+S119P+T416H,

S411V+A393R,

E408R+S386N,

E408R+A425T+S465P+T494A,

L410R+A393R,

S411V+S119P+402F+A393R,

S411V+S119P+402F+A393R+T416H.

In an embodiment the region mutated is the Region: 445-470.

Preferred sub-regions include one or more of: 445-459, 461-470.

Specific preferred positions contemplated include one or more of:

445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470.

Specific mutations include one or more of: G447S, G456C/P, S465P.

Preferred combinations of mutations include one or more of:

G447S+S119P,

S465P+E408R+A425T+T494A.

Increased Specific Activity

In a third aspect the invention relates to a variant of a parentglucoamylase with increased specific activity comprising one or moremutation(s) in the following position(s) or region(s) in the amino acidsequence shown in SEQ ID NO: 2:

Region: 1-18,

Region: 40-62,

Region: 93-127,

Region: 170-184,

Region: 200-212,

Region: 234-246,

Region: 287-319,

Region: 388-414,

and/or in a corresponding position or region in a homologousglucoamylase which displays at least 60% homology with the amino acidsequences shown in SEQ ID NO: 2, with the exception of the followingsubstitutions: S411G.

In an embodiment the region mutated is the Region: 1-18.

Specific preferred positions contemplated include one or more of:

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

Specific mutations are: L3N, I18V.

Preferred combinations of mutations include one or more of:

I18V+T51S+S56A+V59T+L60A.

In an embodiment the region mutated is the Region: 40-62.

Preferred sub-regions include one or more of: 40-47, 58-62.

Specific preferred positions contemplated include one or more of:

40, 41, 42, 43, 44, 45, 46, 47, 49, 51, 53, 56, 58, 59, 60, 61, 62.

Specific mutations include one or more of:

S40C, T43R, T51S/D, T53D, S56A/C, V59T, L60A.

Preferred combinations of mutations include one or more of:

T51S+S56A+V59T+L60A+I18V.

In an embodiment the region mutated is the Region: 93-127.

In an additional embodiment the sub-region is: Region: 93-124

Preferred sub-regions include one or more of: 93-107, 109-111, 113-115.

Specific preferred positions contemplated include one or more of:

93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 109, 110,111, 113, 114, 115, 117, 118, 126, 127.

Specific mutations include one or more of: N93T, P94V, S95N, D97S,L98S/P, S100T/D, A102S/*, N110T, V111P, D112N, E113M/A, T114S, A115Q/G,Y116F, S119A, G127A.

Preferred combinations of mutations include one or more of:

S119P+Y402F,

S119P+Y402F+I189T+Y223F+F227Y.

In an embodiment the region mutated is the Region: 170-184.

Specific mutations include one or more of:

N171R/K/Q/E/W/F/V,

Q172A/R/N/D/C/E/G/H/I/L/K/F/M/P/S/T/W/Y/V,

T173K/R/S/N/Q,

G174A,S,

Y175N/Q/D/E,

D176L,

L177I,

E180N/M,

V181I/T,

N182R/C/E/G/H/I/L/K/M/P/T/W/Y/V, preferred is E,

G183A,

S184 D/N/E/Q/L/I/T/R/K.

In an embodiment the region mutated is the Region: 200-212.

Preferred sub-regions include: 200-211.

Specific preferred positions contemplated include one or more of:

200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211.

Specific mutations include one or more of: A201D, F202L, A203L, T204K,A205R/S, V206L/N, G207N, S208H/T/D, S209T, W212N/A/T.

In an embodiment the region mutated is the Region: 234-246.

Preferred sub-regions include one or more of: 234-240, 242-245.

Specific preferred positions contemplated include one or more of:

234, 235, 236, 237, 238, 239, 240, 242, 243, 244, 245.

In an embodiment the region mutated is the Region: 287-319.

Preferred sub-regions include one or more of: 287-292, 294-301, 313-316.

Specific preferred positions contemplated include one or more of:

287, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300, 301,302, 303, 307, 308, 310, 311, 313, 314, 315, 316, 318, 319.

Specific mutations include one or more of:

S287A/R/N/D/C/Q/E/G/H/I/L/K/M/T/V,

I288L/N/Q,

Y289F,

T290A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/V,

L291I/D/N,

N292D,

D293A/R/N/C/Q/E/G/H/I/L/K/M/S/T/V,

G294A/R/N/D/C/Q/E/H/I/L/K/M/P/S/T/V,

L295A/R/N/D/C/Q/E/G/H/K/M/S/T/V,

S296A/R/N/D/C/Q/E/G/H/I/L/K/M/T/V,

D297A/R/N/C/Q/E/G/H/I/L/K/M/P/S/T/V,

S298A/R/N/D/C/Q/E/G/H/I/L/K/F/M/T/V,

E299A/R/N/D/C/Q/G/H/I/L/K/M/S/T/V.

V301T/I,

A302R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V/, preferred S,

V303T/I,

G304A,

R305A/N/D/C/Q/E/G/H/I/L/F/M/P/S/T/W/Y/V,

Y306A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/T/W/V.

E308A/R/N/D/C/Q/G/H/I/L/K/M/P/S/T/V, preferred Q,

D309L,

T310V/S,

Y311N,

Y312Q/N, preferred is Q,

N313T/S/G, preferred is S,

N315Q/E/R.

In an embodiment the region mutated is the Region: 388-414.

Preferred sub-regions include one or more of: 397-399, 402-406, 412-414.

Specific preferred positions contemplated include one or more of:

388, 389, 390, 394, 397, 398, 399, 402, 403, 404, 405, 406, 409, 412,413, 414.

Specific mutations include one or more of: M398L, S399C/Q/T, Y402F,D403S, S405T, E408C/R, S411V, A412C, D414A.

Preferred combinations of mutations include one or more of:

Y402F+S119P,

Y402F+S119P+I189T+Y223F+F227Y.

In a preferred embodiment of the invention the regions to be mutatedare:

Region: 287-300,

Region: 305-319,

and/or corresponding positions or regions in a homologous glucoamylasewhich displays at least 60% homology with the amino acid sequences shownin SEQ ID NO: 2.

Homology (Identity)

The homology referred to above of the parent glucoamylase is determinedas the degree of identity between two protein sequences indicating aderivation of the first sequence from the second. The homology maysuitably be determined by means of computer programs known in the artsuch as GAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) (Needleman and Wunsch, 1970,Journal of Molecular Biology 48: 443-453). Using Gap with the followingsettings for polypeptide sequence comparison: Gap creation penalty of3.0 and Gap extension penalty of 0.1, the mature part of a polypeptideencoded by an analogous DNA sequence of the invention exhibits a degreeof identity preferably of at least 60%, such as 70%, at least 80%, atleast 90%, more preferably at least 95%, more preferably at least 97%,and most preferably at least 99% with the mature part of the amino acidsequence shown in SEQ ID NO: 2.

Preferably, the parent glucoamylase comprise the amino acid sequences ofSEQ ID NO: 2; or allelic variants thereof; or fragments thereof havingglucoamylase activity.

A fragment of SEQ ID NO: 2 is a polypeptide which has one or more aminoacids deleted from the amino and/or carboxyl terminus of this amino acidsequence. For instance, the AMG G2 (SEQ ID NO: 2) is a fragment of theAspergillus niger G1 glucoamylase (Boel et al., 1984, EMBO J. 3(5):1097-1102) having glucoamylase activity. An allelic variant denotes anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and may result in polymorphism within populations. Gene mutations can besilent (no change in the encoded polypeptide) or may encode polypeptideshaving altered amino acid sequences. An allelic variant of a polypeptideis a polypeptide encoded by an allelic variant of a gene.

The amino acid sequences of homologous parent glucoamylases may differfrom the amino acid sequence of SEQ ID NO: 2 by an insertion or deletionof one or more amino acid residues and/or the substitution of one ormore amino acid residues by different amino acid residues. Preferably,amino acid changes are of a minor nature, that is conservative aminoacid substitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

In another embodiment, the isolated parent glucoamylase is encoded by anucleic acid sequence which hybridises under very low stringencyconditions, preferably low stringency conditions, more preferably mediumstringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with a nucleic acid probewhich hybridises under the same conditions with (i) the nucleic acidsequence of SEQ ID NO: 1, (ii) the cDNA sequence of SEQ ID NO: 1, (iii)a sub-sequence of (i) or (ii), or (iv) a complementary strand of (i),(ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.). The sub-sequence of SEQ ID NO: 1 may be at least 100 nucleotidesor preferably at least 200 nucleotides. Moreover, the sub-sequence mayencode a polypeptide fragment which has glucoamylase activity. Theparent polypeptides may also be allelic variants or fragments of thepolypeptides that have glucoamylase activity.

The nucleic acid sequence of SEQ ID NO: 1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2, or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having glucoamylase activity, from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 35 nucleotides in length. Longer probes canalso be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a polypeptide having glucoamylase. Genomic or otherDNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilised on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO: 1,or sub-sequences thereof, the carrier material is used in a Southernblot. For purposes of the present invention, hybridisation indicatesthat the nucleic acid sequence hybridises to a nucleic acid probecorresponding to the nucleic acid sequence shown in SEQ ID NO: 1 itscomplementary strand, or a sub-sequence thereof, under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridises under these conditions are detected using X-ray film.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridisation, and washing post-hybridization at 5° C. to 10° C. belowthe calculated T_(m) using the calculation according to Bolton andMcCarthy (1962, Proceedings of the National Academy of Sciences USA48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasicphosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standardSouthern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridising a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with the sequenceof SEQ ID NO: 1, or its complementary strand, or a sub-sequence thereof;and (b) isolating the nucleic acid sequence. The sub-sequence ispreferably a sequence of at least 100 nucleotides such as a sequencewhich encodes a polypeptide fragment which has glucoamylase activity.

Contemplated parent glucoamylases have at least 20%, preferably at least40%, more preferably at least 60%, even more preferably at least 80%,even more preferably at least 90%, and most preferably at least 100% ofthe glucoamylase activity of the mature polypeptide of SEQ ID NO: 2.

In a preferred embodiment the variant of the invention has improvedthermal stability and/or increased specific activity, preferably withinthe temperature interval from about 60-80° C., preferably 63-75° C.,preferably at a pH of 4-5, in particular 4.2-4.7, using maltodextrin asthe substrate.

In another preferred embodiment a variant of the invention is used for,e.g., alcohol fermentation.

In a preferred embodiment the parent glucoamylase is the Aspergillusniger G1 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102. Theparent glucoamylase may be a truncated glucoamylase, e.g., the AMG G2glucoamylase.

Cloning a DNA Sequence Encoding a Parent Glucoamylase

The DNA sequence encoding a parent glucoamylase may be isolated from anycell or microorganism producing the glucoamylase in question, usingvarious methods well known in the art. First, a genomic DNA and/or cDNAlibrary should be constructed using chromosomal DNA or messenger RNAfrom the organism that produces the glucoamylase to be studied. Then, ifthe amino acid sequence of the glucoamylase is known, labeledoligonucleotide probes may be synthesized and used to identifyglucoamylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to another known glucoamylase gene couldbe used as a probe to identify glucoamylase-encoding clones, usinghybridization and washing conditions of very low to very highstringency. This is described above.

Yet another method for identifying glucoamylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming glucoamylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for glucoamylase (i.e., maltose),thereby allowing clones expressing the glucoamylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g., thephosphoroamidite method described S. L. Beaucage and M. H. Caruthers,1981, Tetrahedron Letters 22: 1859-1869, or the method described byMatthes et al., 1984, EMBO J. 3: 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesized, e.g., in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or Saikiet al. 1988, Science 239: 487-491.

Site-Directed Mutagenesis

Once a glucoamylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites. In a specificmethod, a single-stranded gap of DNA, the glucoamylase-encodingsequence, is created in a vector carrying the glucoamylase gene. Thenthe synthetic nucleotide, bearing the desired mutation, is annealed to ahomologous portion of the single-stranded DNA. The remaining gap is thenfilled in with DNA polymerase I (Klenow fragment) and the construct isligated using T4 ligase. A specific example of this method is describedin Morinaga et al., 1984, Biotechnology 2: 646-639. U.S. Pat. No.4,760,025 discloses the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into glucoamylase-encoding DNAsequences is described in Nelson and Long, 1989, Analytical Biochemistry180: 147-151. It involves the 3-step generation of a PCR fragmentcontaining the desired mutation introduced by using a chemicallysynthesized DNA strand as one of the primers in the PCR reactions. Fromthe PCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intoan expression plasmid.

Further, Sierks. et al., 1989, Protein Eng. 2: 621-625; Sierks et al.,1990, Protein Eng. 3: 193-198; also describe site-directed mutagenesisin an Aspergillus glucoamylase.

Random Mutagenesis

Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

The random mutagenesis of a DNA sequence encoding a parent glucoamylasemay be conveniently performed by use of any method known in the art.

In relation to the above, a further aspect of the present inventionrelates to a method for generating a variant of a parent glucoamylase,wherein the variant exhibits increased thermal stability relative to theparent, the method comprising:

(a) subjecting a DNA sequence encoding the parent glucoamylase to randommutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a hostcell, and

(c) screening for host cells expressing a glucoamylase variant which hasan altered property (i.e., thermal stability) relative to the parentglucoamylase.

Step (a) of the above method of the invention is preferably performedusing doped primers, as described in the working examples herein (videinfra).

For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) ir-radiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues. When such agents are used, themutagenesis is typically performed by incubating the DNA sequenceencoding the parent enzyme to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

When the mutagenesis is performed by the use of an oligonucleotide, theoligonucleotide may be doped or spiked with the three non-parentnucleotides during the synthesis of the oligonucleotide at the positionswhich are to be changed. The doping or spiking may be done so thatcodons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding theglucoamylase enzyme by any published technique, using, e.g., PCR, LCR orany DNA polymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints. The doping scheme may be made byusing the DOPE program which, inter alia, ensures that introduction ofstop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated ornon-treated gene encoding a parent glucoamylase is subjected to PCRunder conditions that increase the mis-incorporation of nucleotides(Deshler, 1992; Leung et al., 1989, Technique 1: 11-15).

A mutator strain of E. coli (Fowler et al., 1974, Molec. Gen. Genet.133: 179-191), S. cereviseae or any other microbial organism may be usedfor the random mutagenesis of the DNA encoding the glucoamylase by,e.g., transforming a plasmid containing the parent glycosylase into themutator strain, growing the mutator strain with the plasmid andisolating the mutated plasmid from the mutator strain. The mutatedplasmid may be subsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in agenomic or cDNA library prepared from an organism expressing the parentglucoamylase. Alternatively, the DNA sequence may be present on asuitable vector such as a plasmid or a bacteriophage, which as such maybe incubated with or other-wise exposed to the mutagenising agent. TheDNA to be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharboured in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

In some cases it may be convenient to amplify the mutated DNA sequenceprior to performing the expression step b) or the screening step c).Such amplification may be performed in accordance with methods known inthe art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenising agent,the mutated DNA is expressed by culturing a suitable host cell carryingthe DNA sequence under conditions allowing expression to take place. Thehost cell used for this purpose may be one which has been transformedwith the mutated DNA sequence, optionally present on a vector, or onewhich carried the DNA sequence encoding the parent enzyme during themutagenesis treatment. Examples of suitable host cells are thefollowing: gram positive bacteria such as Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encodingfunctions permitting expression of the mutated DNA sequence.

Localized Random Mutagenesis

The random mutagenesis may be advantageously localized to a part of theparent glucoamylase in question. This may, e.g., be advantageous whencertain regions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

The localized, or region-specific, random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

Alternative methods for providing variants of the invention include geneshuffling e.g., as described in WO 95/22625 (from Affymax TechnologiesN.V.) or in WO 96/00343 (from Novo Nordisk NS).

Expression of Glucoamylase Variants

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in enzyme form, using an expression vector whichtypically includes control sequences encoding a promoter, operator,ribosome binding site, translation initiation signal, and, optionally, arepressor gene or various activator genes.

Expression Vector

The recombinant expression vector carrying the DNA sequence encoding aglucoamylase variant of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. The vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated. Examples ofsuitable expression vectors include pMT838.

Promoter

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

Examples of suitable promoters for directing the transcription of theDNA sequence encoding a glucoamylase variant of the invention,especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, thepromoters of the Bacillus licheniformis alpha-amylase gene (amyL), thepromoters of the Bacillus stearothermophilus maltogenic amylase gene(amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase(amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.For transcription in a fungal host, examples of useful promoters arethose derived from the gene encoding A. oryzae TAKA amylase, the TPI(triose phosphate isomerase) promoter from S. cerevisiae (Alber et al.,1982, J. Mol. Appl. Genet. 1: 419-434, Rhizomucor miehei asparticproteinase, A. niger neutral alpha-amylase, A. niger acid stablealpha-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A.oryzae alkaline protease, A. oryzae triose phosphate isomerase or A.nidulans acetamidase.

Expression Vector

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the glucoamylase variantof the invention. Termination and polyadenylation sequences may suitablybe derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

The procedures used to ligate the DNA construct of the inventionencoding a glucoamylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

Host Cells

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of a glucoamylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., an expression vectoras described above in connection with the different types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae.

The host cell may also be a filamentous fungus e.g., a strain belongingto a species of Aspergillus, most preferably Aspergillus oryzae orAspergillus niger, or a strain of Fusarium, such as a strain of Fusariumoxysporium, Fusarium graminearum (in the perfect state named Gribberellazeae, previously Sphaeria zeae, synonym with Gibberella roseum andGibberella roseum f. sp. Cerealis), or Fusarium sulphureum (in theprefect state named Gibberella puricaris, synonym with Fusariumtrichothecioides, Fusarium bactridioides, Fusarium sambucium, Fusariumroseum, and Fusarium roseum var. graminearum), Fusarium cerealis(synonym with Fusarium crokkwellnse), or Fusarium venenatum.

In a preferred embodiment of the invention the host cell is a proteasedeficient or protease minus strain.

This may for instance be the protease deficient strain Aspergillusoryzae JaL 125 having the alkaline protease gene named “alp” deleted.This strain is described in WO 97/35956 (Novo Nordisk).

Filamentous fungi cells may be transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a manner known per se. The use ofAspergillus as a host micro-organism is described in EP 238 023 (NovoNordisk A/S), the contents of which are hereby incorporated byreference.

Method of Producing a Glucoamylase Variant

In a yet further aspect, the present invention relates to a method ofproducing a glucoamylase variant of the invention, which methodcomprises cultivating a host cell under conditions conducive to theproduction of the variant and recovering the variant from the cellsand/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the glucoamylase variant of the invention. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

The glucoamylase variant secreted from the host cells may convenientlybe recovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Starch Conversion

The present invention provides a method of using glucoamylase variantsof the invention for producing glucose and the like from starch.Generally, the method includes the steps of partially hydrolyzingprecursor starch in the presence of alpha-amylase and then furtherhydrolyzing the release of D-glucose from the non-reducing ends of thestarch or related oligo- and polysaccharide molecules in the presence ofglucoamylase by cleaving alpha-1,4 and alpha-1,6 glucosidic bonds.

The partial hydrolysis of the precursor starch utilizing alpha-amylaseprovides an initial breakdown of the starch molecules by hydrolyzinginternal alpha-(1,4)-linkages. In commercial applications, the initialhydrolysis using alpha-amylase is run at a temperature of approximately105° C. A very high starch concentration is processed, usually 30 to 40%solids. The initial hydrolysis is usually carried out for five minutesat this elevated temperature. The partially hydrolyzed starch can thenbe transferred to a second tank and incubated for approximately 1-2 hourat a temperature of 85 to 98° C. to derive a dextrose equivalent (D.E.)of 10 to 15.

The step of further hydrolyzing the release of D-glucose from thenon-reducing ends of the starch or related oligo- and polysaccharidesmolecules in the presence of glucoamylase is normally carried out in aseparate tank at a reduced temperature between 30 and 62° C. Preferablythe temperature of the substrate liquid is dropped to between 55 and 60°C. The pH of the solution is dropped from about 5.5 to 6.5 to a rangebetween 3 and 5.5. Preferably, the pH of the solution is 4 to 4.5. Theglucoamylase is added to the solution and the reaction is carried outfor 24-72 hours, preferably 36-48 hours.

By using a thermostable glucoamylase variant of the inventionsaccharification processes may be carried out at a higher temperaturethan traditional batch saccharification processes. According to theinvention saccharification may be carried out at temperatures in therange from above 60-80° C., preferably 63-75° C. This apply both fortraditional batch processes (described above) and for continuoussaccharification processes.

Actually, continuous saccharification processes including one or moremembrane separation steps, i.e., filtration steps, must be carried outat temperatures of above 60° C. to be able to maintain a reasonably highflux over the membrane or to minimize microbial contamination.Therefore, the thermostable variants of the invention provides thepossibility of carrying out large scale continuous saccharificationprocesses at a fair price and/or at a lower enzyme protein dosage withina period of time acceptable for industrial saccharification processes.According to the invention the saccharification time may even beshortened.

The activity of the glucoamylase variant (e.g., AMG variant) of theinvention is generally substantially higher at temperatures between60-80° C. than at the traditionally used temperature between 30-60° C.Therefore, by increasing the temperature at which the glucoamylaseoperates the saccharification process may be carried out within ashorter period of time.

Further, by improving the thermal stability the T_(1/2) (half-time, asdefined in the “Materials and Methods” section) is improved. As thethermal stability of the glucoamylase variants of the invention isimproved a minor amount of glucoamylase need to be added to replace theglucoamylase being inactivated during the saccharification process. Moreglucoamylase is maintained active during saccharification processaccording to the present invention. Furthermore, the risk of microbialcontamination is also reduced when carrying the saccharification processat temperature above 63° C.

The glucose yield from a typical saccharification trial withglucoamylase, acid amylase and pullulanase is 95.5-96.5%. The remainingcarbohydrates typically consist of 1% maltose, 1.5-2% isomaltose and1-1.5% higher oligosaccharides. The disaccharides are produced since theglucoamylase at high concentrations of glucose and high dry-solid levelshas a tendency to form reversion products.

A glucoamylase with an increased specific activity towards saccharidespresent in the solution after liquefaction and saccharides formed duringsaccharification would be an advantage as a reduced enzyme proteindosage or a shorter process time then could be used. In general, theglucoamylase has a preference for substrates consisting of longersaccharides compared to short chain saccharides and the specificactivity towards e.g., maltoheptaose is therefore approximately 6 timeshigher than towards maltose. An increased specific activity towardsshort chain saccharides such as maltose (without reducing the activitytowards oligosaccharides) would therefore also permit using a lowerenzyme dosage and/or shorter process time.

Furthermore, a higher glucose yield can be obtained with a glucoamylasevariant with an increased alpha-1,4 hydrolytic activity (if thealpha-1,6 activity is unchanged or even decreased), since a reducedamount of enzyme protein is being used, and alpha-1,6 reversion productformation therefore is decreased (less isomaltose).

The specific activity may be measured using the method described in the“Materials & Methods” section at 37° C. or 60° C.

Example of saccharification process wherein the glucoamylase variants ofthe invention may be used include the processes described in JP3-224493; JP 1-191693; JP 62-272987; EP 452,238, and WO 99/27124 (allreferences are hereby incorporated by reference).

In a further aspect the invention relates to a method of saccharifying aliquefied starch solution, comprising the steps

(a) a saccharification step during which step one or more enzymaticsaccharification stages takes place, and the subsequent step of

(b) one or more high temperature membrane separation steps

wherein the enzymatic saccharification is carried out using athermostable glucoamylase variant of the invention.

The glucoamylase variant(s) of the invention may be used in the presentinventive process in combination with an enzyme that hydrolyzes onlyalpha-1,6-glucosidic bonds in molecules with at least four glucosylresidues. Preferentially, the glucoamylase variant of the invention canbe used in combination with pullulanase or isoamylase. The use ofisoamylase and pullulanase for debranching, the molecular properties ofthe enzymes, and the potential use of the enzymes with glucoamylase isset forth in G. M. A. van Beynum et al., Starch Conversion Technology,Marcel Dekker, New York, 1985, 101-142.

In a further aspect the invention relates to the use of a glucoamylasevariant of the invention in a starch conversion process.

Further, the glucoamylase variant of the invention may be used in acontinuous starch conversion process including a continuoussaccharification step.

The glucoamylase variants of the invention may also be used inimmobilised form. This is suitable and often used for producingspeciality syrups, such as maltose syrups, and further for the raffinatestream of oligosaccharides in connection with the production of fructosesyrups.

The glucoamylase of the invention may also be used in a process forproducing ethanol for fuel or beverage or may be used in a fermentationprocess for producing organic compounds, such as citric acid, ascorbicacid, lysine, glutamic acid.

Materials & Methods

Materials:

Enzymes:

AMG G1: Aspergillus niger glucoamylase G1 disclosed in Boel et al.,1984, EMBO J. 3(5): 1097-1102, and SEQ ID NO: 13, available from NovoNordisk.

AMG G2: Truncated Aspergillus niger glucoamylase G1 shown in SEQ ID NO:2, available from Novo Nordisk)

Solutions:

Buffer: 0.05 M sodium acetate (6.8 g in 1 l milli-Q-water), pH 4.5

Stop solution: 0.4 M NaOH

GOD-perid, 124036, Boehringer Mannheim

Substrate:

Maltose: 29 mM (1 g maltose in 100 ml 50 mM sodium acetate, pH 4.5)(Sigma)

Maltoheptaose: 10 mM, 115 mg/10 ml (Sigma)

Host Cell:

A. oryzae JaL 125: Aspergillus oryzae IFO 4177 available from Institutefor Fermention, Osaka; 17-25 Juso Hammachi 2-Chome Yodogawa-ku, Osaka,Japan, having the alkaline protease gene named “alp” (described byMurakami et al., 1991, Agric. Biol. Chem. 55: 2807-2811) deleted by aone step gene replacement method (described by G. May in “AppliedMolecular Genetics of Filamentous Fungi” (1992), p. 1-25. Eds. J. R.Kinghorn and G. Turner; Blackie Academic and Professional), using the A.oryzae pyrG gene as marker. Strain JaL 125 is further disclosed in WO97/35956 (Novo Nordisk).

Microorganisms:

Strain: Saccharomyces cerevisiae YNG318: MATαleu2-Δ2 ura3-52 his4-539pep4-Δ1[cir+]

Plasmids:

pCAMG91: see FIG. 1. Plasmid comprising the Aspergillus niger G1glucoamylase (AMG G1). The construction of pCAMG91 is described in Boelet al., 1984, EMBO J. 3(7):1581-1585. pMT838: Plasmid encoding thetruncated Aspergillus niger glucoamylase G2 (SEQ ID NO: 2). pJSO026 (S.cerevisiae expression plasmid) (J. S. Okkels, (1996) “A URA3-promoterdeletion in a pYES vector increases the expression level of a fungallipase in Saccharomyces cerevisiae. Recombinant DNA Biotechnology III:The Integration of Biological and Engineering Sciences, vol. 782 of theAnnals of the New York Academy of Sciences) More specifically, theexpression plasmid pJSO37, is derived from pYES 2.0 by replacing theinducible GAL1-promoter of pYES 2.0 with the constitutively expressedTPI (triose phosphate isomerase)-promoter from Saccharomyces cerevisiae(Albert and Karwasaki, 1982, J. Mol. Appl. Genet. 1: 419-434), anddeleting a part of the URA3 promoter.

Methods:

Transformation of Saccharomyces cerevisiae YNG318

The DNA fragments and the opened vectors are mixed and transformed intothe yeast Saccharomyces cerevisiae YNG318 by standard methods.

Determining Specific Activity As k_(cat) (sec.⁻¹)

750 microL substrate (1% maltose, 50 mM Sodium acetat, pH 4.3) isincubated 5 minutes at selected temperature, such as 37° C. or 60° C.

50 microL enzyme diluted in sodium acetate is added.

Aliquots of 100 microL are removed after 0, 3, 6, 9 and 12 minutes andtransferred to 100 microL 0.4 M Sodium hydroxide to stop the reaction. Ablank is included.

20 microL is transferred to a Micro titre plates and 200 microLGOD-Perid solution is added. Absorbance is measured at 650 nm after 30minutes incubation at room temperature. Glucose is used as standard andthe specific activity is calculated as k_(cat) (sec.⁻¹).

Determination of AGU Activity and As AGU/mg

One Novo Amyloglucosidase Unit (AGU) is defined as the amount of enzymewhich hydrolyzes 1 micromole maltose per minute at 37° C. and pH 4.3. Adetailed description of the analytical method (AEL-SM-0131) is availableon request from Novo Nordisk.

The activity is determined as AGU/ml by a method modified after(AEL-SM-0131) using the Glucose GOD-Perid kit from Boehringer Mannheim,124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml.

375 microL substrate (1% maltose in 50 mM Sodium acetate, pH 4.3) isincubated 5 minutes at 37° C. 25 microL enzyme diluted in sodium acetateis added. The reaction is stopped after 10 minutes by adding 100 microL0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and200 microL GOD-Perid solution is added. After 30 minutes at roomtemperature, the absorbance is measured at 650 nm and the activitycalculated in AGU/ml from the AMG-standard.

The specific activity in AGU/mg is then calculated from the activity(AGU/ml) divided with the protein concentration (mg/ml).

Transformation of Aspergillus oryzae (General Procedure)

100 ml of YPD (Sherman et al., 1981, Methods in Yeast Genetics, ColdSpring Harbor Laboratory) are inoculated with spores of A. oryzae andincubated with shaking for about 24 hours. The mycelium is harvested byfiltration through miracloth and washed with 200 ml of 0.6 M MgSO₄. Themycelium is suspended in 15 ml of 1.2 M MgSO₄, 10 mM NaH₂PO₄, pH 5.8.The suspension is cooled on ice and 1 ml of buffer containing 120 mg ofNovozym™ 234 is added. After 5 min., 1 ml of 12 mg/ml BSA (Sigma typeH25) is added and incubation with gentle agitation continued for 1.5-2.5hours at 37° C. until a large number of protoplasts is visible in asample inspected under the microscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayed with 5 ml of 0.6 M sorbitol, 100 mMTris-HCl, pH 7.0. Centrifugation is performed for 15 min. at 1000 g andthe protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 min. at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated. Finally, the protoplasts areresuspended in 0.2-1 ml of STC.

100 microliters of protoplast suspension are mixed with 5-25 μg of p3SR2(an A. nidulans amdS gene carrying plasmid described in Hynes et al.,1983, Mol. and Cel. Biol. 3(8): 1430-1439) in 10 microliters of STC. Themixture is left at room temperature for 25 min. 0.2 ml of 60% PEG 4000(BDH 29576), 10 mM CaCl₂ and 10 mM Tris-HCl, pH 7.5 is added andcarefully mixed (twice) and finally 0.85 ml of the same solution areadded and carefully mixed. The mixture is left at room temperature for25 min., spun at 2.500 g for 15 min. and the pellet is resuspended in 2ml of 1.2 M sorbitol. After one more sedimentation the protoplasts arespread on minimal plates (Cove, 1966, Biochem. Biophys. Acta 113: 51-56)containing 1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogen source and20 mM CsCl to inhibit background growth. After incubation for 4-7 daysat 37° C. spores are picked, suspended in sterile water and spread forsingle colonies. This procedure is repeated and spores of a singlecolony after the second re-isolation are stored as a definedtransformant.

Fed Batch Fermentation

Fed batch fermentation is performed in a medium comprising maltodextrinas a carbon source, urea as a nitrogen source and yeast extract. The fedbatch fermentation is performed by inoculating a shake flask culture ofA. oryzae host cells in question into a medium comprising 3.5% of thecarbon source and 0.5% of the nitrogen source. After 24 hours ofcultivation at pH 5.0 and 34° C. the continuous supply of additionalcarbon and nitrogen sources are initiated. The carbon source is kept asthe limiting factor and it is secured that oxygen is present in excess.The fed batch cultivation is continued for 4 days, after which theenzymes can be recovered by centrifugation, ultrafiltration, clearfiltration and germ filtration.

Purification

The culture broth is filtrated and added ammoniumsulphate (AMS) to aconcentration of 1.7 M AMS and pH is adjusted to pH 5. Precipitatedmaterial is removed by centrifugation and the solution containingglucoamylase activity is applied on a Toyo Pearl Butyl column previouslyequilibrated in 1.7 M AMS, 20 mM sodium acetate, pH 5. Unbound materialis washed out with the equilibration buffer. Bound proteins are elutedwith 10 mM sodium acetate, pH 4.5 using a linear gradient from 1.7-0 MAMS over 10 column volumes. Glucoamylase containing fractions arecollected and dialysed against 20 mM sodium acetate, pH 4.5. Thesolution was then applied on a Q sepharose column, previouslyequilibrated in 10 mM piperazin, Sigma, pH 5.5. Unbound material iswashed out with the equilibration buffer. Bound proteins are eluted witha linear gradient of 0-0.3 M Sodium chloride in 10 mM piperazin, pH 5.5over 10 column volumes. Glucoamylase containing fractions are collectedand the purity was confirmed by SDS-PAGE.

T_(1/2) (Half-Life) Method I

The thermal stability of variants is determined as T_(1/2) using thefollowing method: 950 microliter 50 mM sodium acetate buffer (pH 4.3)(NaOAc) is incubated for 5 minutes at 68° C. or 70° C. 50 microlitersenzyme in buffer (4 AGU/ml) is added. 2×40 microliter samples are takenat 0, 5, 10, 20, 30 and 40 minutes and chilled on ice. The activity(AGU/ml) measured before incubation (0 minutes) is used as reference(100%). The decline in stability (in percent) is calculated as afunction of the incubation time. The % residual glucoamylase activity isdetermined at different times. T_(1/2) is the period of time until whichthe % relative activity is decreased to 50%.

T_(1/2) (Half-Life) (Method II)

The T_(1/2) is measured by incubating the enzyme (ca 0.2 AGU/ml) inquestion in 30% glucose, 50 mM Sodium acetate at pH 4.5 at thetemperature in question (e.g., 70° C.). Samples are withdrawn at settime intervals and chilled on ice and residual enzyme activity measuredby the pNPG method (as described below).

The % residual glucoamylase activity is determined at different times.T_(1/2) is the period of time until which the % relative activity isdecreased to 50%.

Residual Enzyme Activity (pNPG Method)

pNPG Reagent:

0.2 g pNPG (p-nitrophenylglucopyranoside) is dissolved in 0.1 M acetatebuffer (pH 4.3) and made up to 100 ml.

Borate Solution:

3.8 g Na₂B₄O₇10H₂O is dissolved in Milli-Q water and made up to 100 ml.

25 microL samples are added 50 microL substrate and incubated 2 hr at50° C. The reaction is stopped by adding 150 micoL ml borate solution.The optical density is measured at 405 nm, and the residual activitycalculated.

Construction of pAMGY

The pAMGY vector was constructed as follows: The lipase gene in pJSO026was replaced by the AMG gene, which was PCR amplified with the forwardprimer; FG2: 5′-CAT CCC CAG GAT CCT TAC TCA GCA ATG-3′ (SEQ ID NO: 10)and the reverse primer: RG2: 5′-CTC AAA CGA CTC ACC AGC CTC TAG AGT-3′(SEQ ID NO: 11) using the template plasmid pLAC103 containing the AMGgene. The pJSO026 plasmid was digested with XbaI and SmaI at 37° C. for2 hours and the PCR amplicon was blunt ended using the Klenow fragmentand then digested with XbaI. The vector fragment and the PCR ampliconwere ligated and transformed into E. coli by electrotransformation. Theresulting vector is designated pAMGY.

Construction of pLaC103

The A. niger AMGII cDNA clone (Boel et al., 1984, supra) is used assource for the construction of pLaC103 aimed at S. cerevisiae expressionof the GII form of AMG.

The construction takes place in several steps, out lined below.

pT7-212 (EP 37856/U.S. Pat. No. 5,162,498) is cleaved with XbaI,blunt-ended with Klenow DNA polymerase and dNTP. After cleavage withEcoRI the resulting vector fragment is purified from an agarosegel-electrophoresis and ligated with the 2.05 kb EcoR1-EcoRV fragment ofpBoel53, thereby recreating the XbaI site in the EcoRV end of the AMGencoding fragment in the resulting plasmid pG2x.

In order to remove DNA upstream of the AMG cds, and furnish the AMGencoding DNA with an appropriate restriction endonuclease recognitionsite, the following construct was made:

The 930 by EcoRI-PstI fragment of p53 was isolated and subjected to AluIcleavage, the resulting 771 by Alu-PstI fragment was ligated into pBR322with blunt-ended EcoRI site (see above) and cleaved with PstI In theresulting plasmid pBR-AMG′, the EcoRI site was recreated just 34 by fromthe initiation codon of the AMG cds.

From pBR-AMG′ the 775 by EcoRI-PstI fragment was isolated and joinedwith the 1151 by PstI-XbaI fragment from pG2x in a ligation reactionincluding the XbaI-EcoRI vector fragment of pT7-212.

The resulting plasmid pT7GII was submitted to a BamHI cleavage inpresence of alkaline phosphatase followed by partial SphI cleavage afterinactivation of the phosphatase. From this reaction was the 2489 bySphI-BamHI fragment, encompassing the S.c. TPI promoter linked to theAMGII cds.

The above fragment together with the 1052 by BamHI fragment of pT7GIIwas ligated with the alkaline phosphatase treated vector fragment ofpMT743 (EP 37856/U.S. Pat. No. 5,162,498), resulting from SphI-BamHIdigestion. The resulting plasmid is pLaC103.

Screening for Thermostable AMG Variants

The libraries are screened in the thermostable filter assay describedbelow.

Filter Assay for Thermostability

Yeast libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on SCFura-agarplates with 100 micrograms/ml ampicillin at 30° C. for at least 72hours. The colonies are replica plated to PVDF filters (Immobilon-P,Millipore, Bedford) activated with methanol for 1 min or alternatively aProtran filter (no activation) and subsequently washed in 0.1 M NaAc andthen incubated at room temperature for 2 hours. Colonies are washed fromPVDF/Protran filters with tap water. Each filter sandwiches andPVDF/Protran filters are specifically marked with a needle beforeincubation in order to be able to localize positive variants on thefilters after the screening. The PVDF filters with bound variants aretransferred to a container with 0.1 M NaAc, pH 4.5 and incubated at 47°C. or alternatively 67-69° C. in case of Protran filters for 15 minutes.The sandwich of cellulose acetate and nitrocellulose filters on SCura-agar plates are stored at room temperature until use. Afterincubation, the residual activities are detected on plates containing 5%maltose, 1% agarose, 50 mM NaAc, pH 4.5. The assay plates with PVDFfilters are marked the same way as the filter sandwiches and incubatedfor 2 hours at 50° C. After removal of the PVDF filters, the assayplates are stained with Glucose GOD perid (Boehringer Mannheim GmbH,Germany). Variants with residual activity are detected on assay platesas dark green spots on white background. The improved variants arelocated on the storage plates. Improved variants are rescreened twiceunder the same conditions as the first screen.

General Method for Random Mutagenesis by Use of the DOPE Program

The random mutagenesis may be carried out by the following steps:

1. Select regions of interest for modification in the parent enzyme,

2. Decide on mutation sites and non-mutated sites in the selectedregion,

3. Decide on which kind of mutations should be carried out, e.g., withrespect to the desired stability and/or performance of the variant to beconstructed,

4. Select structurally reasonable mutations,

5. Adjust the residues selected by step 3 with regard to step 4.

6. Analyze by use of a suitable dope algorithm the nucleotidedistribution.

7. If necessary, adjust the wanted residues to genetic code realism,e.g., taking into account constraints resulting from the genetic code,e.g., in order to avoid introduction of stop codons; the skilled personwill be aware that some codon combinations cannot be used in practiceand will need to be adapted

8. Make primers

9. Perform random mutagenesis by use of the primers

10. Select resulting glucoamylase variants by screening for the desiredimproved properties.

Dope Algorithm

Suitable dope algorithms for use in step 6 are well known in the art.One such algorithm is described by Tomandl et al., 1997, Journal ofComputer-Aided Molecular Design 11: 29-38. Another algorithm is DOPE(Jensen et al., 1998, Nucleic Acids Research 26: 697-702).

Method of Extracting Important Regions for Temperature Activity UsingMolecular Simulation

The X-ray structure and/or the model-build structure of the enzyme ofinterest, here AMG, are subjected to molecular dynamics simulations. Themolecular dynamics simulation are made using the CHARMM (from Molecularsimulations (MSI)) program or other suitable programs, e.g., DISCOVER(from MSI). The dynamics are made in vacuum, or including crystalwaters, or with the enzyme in question embedded in a suitable water,e.g., a sphere or a box. The simulation are run for 300 picoseconds (ps)or more, e.g., 300-1200 ps. The isotropic fluctuations are extracted forthe CA carbons of the structures and a comparison between the structuresis made. More details on how to get the isotropic fluctuations can befound in the CHARMM manual (available from MSI) and hereby incorporatedherein by reference.

The molecular dynamics simulation can be carried out using standardcharges on the chargeable amino acids. For instance, Asp and Glu isnegatively charged and Lys and Arg are positively charged. Thiscondition resembles the medium pH of approximately 7.0. To analyze alower pH, titration of the molecule can be done to obtain the alteredpKa's of the normal titrateable residues within pH 2-10; Lys, Arg, Asp,Glu, Tyr and His. Also Ser, Thr and Cys are titrateable but are nottaking into account here. Here the altered charges due to the pH hasbeen described as all Arg, Lys negative at high pH, and all Asp, Glu areuncharged. This imitates a pH around 4 to 5 where the titration Asp andGlu normally takes place.

Model building of the enzyme of interest can be obtained by using theHOMOLOGY model in the MSI program package. The crystal structure ofAspergillus awamori variant X100 can be found in, e.g., 3GLY and 1DOG inthe Brookhaven database.

EXAMPLES Example 1 Construction of AMG G2 Variants

Site-Directed Mutagenesis:

For the construction of variants of AMG G2 (SEQ ID NO: 2) the commercialkit, Chameleon double-stranded, site-directed mutagenesis kit was usedaccording to the manufacturer's instructions.

The gene encoding the AMG G2 enzyme in question is located on pMT838prepared by deleting the DNA between G2 nt. 1362 and G2 nt. 1530 inplasmid pCAMG91 (see FIG. 1) comprising the AMG G1 form.

In accordance with the manufacturer's instructions the ScaI site of theAmpicillin gene of pMT838 was changed to a MluI site by use of thefollowing primer:

7258: 5′ p gaa tga ctt ggt tga cgc gtc acc agt cac 3′ (SEQ ID NO: 3).

(Thus changing the ScaI site found in the ampicillin resistance gene andused for cutting to a MluI site). The pMT838 vector comprising the AMGgene in question was then used as a template for DNA polymerase andoligo 7258 (SEQ ID NO: 3) and 21401 (SEQ ID NO: 4).

Primer no. 21401 (SEQ ID NO: 4) was used as the selection primer.

21401: 5′ p gg gga tca tga tag gac tag cca tat taa tga agg gca tat accacg cct tgg acc tgc gtt ata gcc 3′

(Changes the ScaI site found in the AMG gene without changing the aminoacid sequence).

The desired mutation (e.g., the introduction of a cystein residue) isintroduced into the AMG gene in question by addition of appropriateoligos comprising the desired mutation.

The primer 107581 was used to introduce T12P

107581: 5′ pgc aac gaa gcg ccc gtg get cgt ac 3′ (SEQ ID NO: 5)

The mutations are verified by sequencing the whole gene. The plasmid wastransformed into A. oryzae using the method described above in the“Materials & Methods” section. The variant was fermented and purified asdescribed above in the “Materials & Methods” section.

Example 2 Construction, by Localized Random, Doped Mutagenesis, of A.niger AMG Variants Having Improved Thermostability Compared to theParent Enzyme

To improve the thermostability of the A. niger AMG random mutagenesis inpre-selected region was performed.

Residue:

Region: L19-G35

Region: A353-V374

The DOPE software (see Materials and Methods) was used to determinespiked codons for each suggested change in the above regions minimizingthe amount of stop codons (see table 1). The exact distribution ofnucleotides was calculated in the three positions of the codon to givethe suggested population of amino acid changes. The doped regions weredoped specifically in the indicated positions to have a high chance ofgetting the desired residues, but still allow other possibilities.

The first column is the amino acid to be mutated, the second column isthe percentage of wild type and the third column defined the new aminoacid(s).

TABLE 1 Doping in L19-G35 L19 90% N N20 95% T N21 Constant I22 ConstantG23 95% A A24 90% S, T D25 93% S, T, R G26 95% A A27 90% S, T W28 <80%  R, Y V29 Constant S30 93% T, N G31 95% A A32 95% V D33 80% R, K, H S3490% N G35 Constant

The resulting doped oligonucleotide strand is shown in table 2 as sensestrand: with the primer sequence, the wild type nucleotide sequence, theparent amino acid sequence and the distribution of nucleotides for eachdoped position.

TABLE 2 Position: 19 20 21 22 23 24 25 26 27 A.a. seq.: L N N I G A D GA primer: 12T A3T AAC ATC G4G 5CG 67C G4T 8CT wt. seq.: CTG AAT AAC ATCGGG GCG GAC GGT GCT Pos. (cont.): 28 29 30 31 32 33 34 35 A.a. (cont.):W V S G A D S G primer: 91010 GTG 1112C G4C G13G 141516 1718T GGCWt seq.: TGG GTG TCG GGC GCG GAC TCT GGC

Distribution of nucleotides for each doped position.

1: A10, C90

2: A6, T94

3: A95, C5

4: G95, C5

5: G91, A3, T3, C3

6: G95, A3, C2

7: G3, A95, C2

8: G92, A4, T4

9: A3, T97

10: G95, T5

11: G3, A97

12: G95, A2, C3

13: T5, C95

14: G88, A8, C4

15: G7, A93

16: G4, C96

17: G4, A96

18: G95, A2, C3

Forward primer (SEQ ID NO: 6):FAMGII ′5-C GAA GCG ACC GTG GCT CGT ACT GCC ATC12T A3T AAC ATC G4G 5CG 67C G4T 8CT 91010 GTG1112C G4C G13G 141516 1718T GGC ATT GTC GTT GCTAGT CCC AGC ACG GAT AAC-3′ Reverse primer (SEQ ID NO: 7):RAMG1: 5′-GAT GGC AGT ACG AGC CAC GGT CGC TTC G-3′

TABLE 3 Doping in region A353-V374: A353 <80%   D, E, Q, N, Y L354 90%Q, E Y355 90% N, Q S356 90% T, D, N G357 80% P, A, S, T A358 93% S A35990% S, T, N T360 90% R, K G361 85% A, S, T T362 90% S Y363 Constant S36493% D S365 93% N, Q, K S366 93% P, D S367 Constant S368 93% D, N, T T36993% Q, E Y370 Constant S371 93% N S372 93% N, T I373 Constant V374 93%N, Y, H

The resulting doped oligonucleotide strand is shown in table 4 as sensestrand: with the primer sequence, wild type nucleotide sequence, theparent amino acid sequence and the distribution of nucleotides for eachdoped position.

TABLE 4 Position: 353 354 355 356 357 358 359 360 361 362 363 A.a. seq.:A L Y S D A A T G T Y primer: 123 45A 6AC 78C 910T 11CT 1213T 1415A1617C 18CC TAC Wt. seq.: GCA CTG TAC AGC GAT GCT GCT ACT GGC ACC TACPos. (cont.): 364 365 366 367 368 369 370 371 372 373 374A.a. seq. (cont.): S S S S S T Y S S I V primer (cont.): 1920T A21222324C AGT 1425C 2627G T28T A16T 2930T ATT 313233 wt. Seq. (cont.): TCTTCG TCC AGT TCG ACT TAT AGT AGC ATT GTA

Distribution of nucleotides for each doped position.

1: G91, A3, T3, C3

2: A13, C87

3: A40, T60

4: G3, A3, C94

5: A6, T94

6: G4, A4, T92

7: G2, A96, C2

8: G93, A3.5, C3.5

9: G87, A8, C5

10: A84, C16

11: G93, T7

12: G92, A5, T3

13: A3, C97

14: G3, A97

15: G2, A2, T4, C92

16: G93, A7

17: G93, C7

18: A90, T10

19: G4, A96

20: G95, A5

21: G96, A4

22: G3, C97

23: G2, A1, T95, C2

24: A3, C97

25: G95, A3, C2

26: G2, A96, C2

27: A5, C95

28: A95, T5

29: G2, A98

30: G94, A4, C2

31: G94, A3, T1, C2

32: A4, T96

33: A20, C80

Primer: FAMGIV (SEQ ID NO: 8)5′-GTG TCG CTG GAC TTC TTC AAG 123 45A 6AC 78C910T 11CT 1213T 1415A 1617C 18CC TAC 1920T A21222324C AGT 1425C 2627G T28T A16T 2930C ATT 313233GAT GCC GTG AAG ACT TTC GCC GA-3′ Primer RAMGVI (SEQ ID NO: 9)5′-ctt gaa gaa gtc cag cga cac-3′Random Mutagenesis

The spiked oligonucleotides apparent from Tables 2 and 3 (which by acommon term is designated FAMG) and reverse primers RAMG for the L19-G35region and specific SEQ ID NO: 2 primers covering the N-terminal (FG2:5′-CAT CCC CAG GAT CCT TAC TCA GCA ATG-3′ (SEQ ID NO: 10) and C-terminal(RG2: 5″-CTC AAA CGA CTC ACC AGC CTC TAG AGT (SEQ ID NO: 11) are used togenerate PCR-library-fragments by the overlap extension method (Hortonet al., 1989, Gene 77: 61-68) with an overlap of 21 base pairs. PlasmidpAMGY is template for the Polymerase Chain Reaction. The PCR fragmentsare cloned by homologous recombination in the E. coli/yeast shuttlevector pAMGY (see Materials and Methods).

Screening

The library was screened in the thermostability filter assays using aProtran filter and incubating at 67-69° C. as described in the “Material& Methods” section above

Example 3 Construction, by PCR Shuffling Spiked with DNA Oligos, of A.niger AMG Variants Having Improved Thermostability Compared to theParent Enzyme

The polymerase chain reaction (PCR) method was used to prepare DNAfragments carrying the AMG gene and flanking regions. Approximately 10ug DNA was digested with Dnase, and run on a 2% agarose gel. Fragmentsof 50-150 by were purified from the gel. Approximately 1 ug purifiedfragments were mixed with a 5-15 fold molar excess of oligos carryingthe desired mutations. The oligos were of the following kind (for theconstruction of Hklib1, Hklib2, Hklib3 etc., respectively):

Hklib1: Hk1-T2X: (SEQ ID NO: 14)5′-ATGTGATTTCCAAGCGCGCGVNNTTGGATTCATGGTTGAGCAA Hk1-N9X: (SEQ ID NO: 15)5′-CCTTGGATTCATGGTTGAGCVNNGAAGCGACCGTGGCTCGTAC Hk1-A11X: (SEQ ID NO: 16)5′-ATTCATGGTTGAGCAACGAAVNNACCGTGGCTCGTACTGCCAT Hk1-L66X: (SEQ ID NO: 17)5′-TCCTCAAGACCCTCGTCGATVNNTTCCGAAATGGAGATACCAG Hk1-S386X:(SEQ ID NO: 18) 5′-CTTTCGCCGATGGCTTCGTCVNNATTGTGGAAACTCACGCCGCHk1-E389X: (SEQ ID NO: 19)5′-ATGGCTTCGTCTCTATTGTGVNNACTCACGCCGCAAGCAACGG Hk1-T390X:(SEQ ID NO: 20) 5′-GCTTCGTCTCTATTGTGGAAVNNCACGCCGCAAGCAACGGCTCHk1-A393X: (SEQ ID NO: 21)5′-CTATTGTGGAAACTCACGCCVNNAGCAACGGCTCCATGTCCGA Hk1-S394X:(SEQ ID NO: 22) 5′-TTGTGGAAACTCACGCCGCAVNNAACGGCTCCATGTCCGAGCAHk1-N395X: (SEQ ID NO: 23)5′-TGGAAACTCACGCCGCAAGCVNNGGCTCCATGTCCGAGCAATA Hk1-G396X:(SEQ ID NO: 24) 5′-AAACTCACGCCGCAAGCAACVNNTCCATGTCCGAGCAATACGAHk1-K404X: (SEQ ID NO: 25)5′-CCATGTCCGAGCAATACGACVNNTCTGATGGCGAGCAGCTTTC Hk1-D406X:(SEQ ID NO: 26) 5′-CCGAGCAATACGACAAGTCTVNNGGCGAGCAGCTTTCCGCTCGHk1-E408X: (SEQ ID NO: 27)5′-AATACGACAAGTCTGATGGCVNNCAGCTTTCCGCTCGCGACCT Hk1-L410X:(SEQ ID NO: 28) 5′-ACAAGTCTGATGGCGAGCAGVNNTCCGCTCGCGACCTGACCT Hk1-L423X:(SEQ ID NO: 29) 5′-CCTGGTCTTATGCTGCTCTGVNNACCGCCAACAACCGTCGTAAHk1-N426X: (SEQ ID NO: 30)5′-ATGCTGCTCTGCTGACCGCCVNNAACCGTCGTAACTCCGTCGTG Hk1-N427X:(SEQ ID NO: 31) 5′-CTGCTCTGCTGACCGCCAACVNNCGTCGTAACTCCGTCGTGCCTHk1-Y402X: (SEQ ID NO: 32)5′-ACGGCTCCATGTCCGAGCAANNCGACAAGTCTGATGGCGAGCAGCT Hklib2:Hk2-L234X-SENSE: (SEQ ID NO: 33)5′-CTGGACCGGCAGCTTCATTNNKGCCAACTTCGATAGCAGCC Hk2-A235S-ANTISENSE:(SEQ ID NO: 34) 5′-GAACGGCTGCTATCGAAGTTAGACAGAATGAAGCTGCCGGTCHk2-F237X-SENSE: (SEQ ID NO: 35)5-CAGCTTCATTCTGGCCAACNATGATAGCAGCCGTTCCGGCA Hk2-D238T-ANTISENSE:(SEQ ID NO: 36) 5′-CCTTGCCGGAACGGCTGCTAGTGAAGTTGGCCAGAATGAAGCHk2-D238S-ANTISENSE: (SEQ ID NO: 37)5′-CCTTGCCGGAACGGCTGCTAGAGAAGTTGGCCAGAATGAAGC Hk2-S239X-SENSE:(SEQ ID NO: 38) 5′-TCATTCTGGCCAACTTCGATNNCAGCCGTTCCGGCAAGGACGHk2-S240G-ANTISENSE: (SEQ ID NO: 39)5′-TTGCGTCCTTGCCGGAACGACCGCTATCGAAGTTGGCCAGAA Hk2-S242X-ANTISENSE:(SEQ ID NO: 40) 5′-GGGTGTTTGCGTCCTTGCCAKNACGGCTGCTATCGAAGTTGHk2-G243X-ANTISENSE: (SEQ ID NO: 41)5′-GGAGGGTGTTTGCGTCCTTAKNGGAACGGCTGCTATCGAAG Hk2-K244R-SENSE:(SEQ ID NO: 42) 5′-CGATAGCAGCCGTTCCGGCAGAGACGCAAACACCCTCCTGGHk2-T310V-ANTISENSE: (SEQ ID NO: 43)5′-ACGGGTTGCCGTTGTAGTAAACGTCCTCAGGGTACCGACCC Hk2-T310S-ANTISENSE:(SEQ ID NO: 44) 5′-ACGGGTTGCCGTTGTAGTAAGAGTCCTCAGGGTACCGACCCHk2-Y311N-SENSE: (SEQ ID NO: 45)5′-TCGGTACCCTGAGGACACGAATTACAACGGCAACCCGTGGT Hk2-Y312Q-ANTISENSE:(SEQ ID NO: 46) 5′-GGAACCACGGGTTGCCGTTTTGGTACGTGTCCTCAGGGTACHk2-Y312N-ANTISENSE: (SEQ ID NO: 47)5′-GGAACCACGGGTTGCCGTTATTGTACGTGTCCTCAGGGTAC Hk2-N313T-SENSE:(SEQ ID NO: 48) 5′-CCCTGAGGACACGTACTACACTGGCAACCCGTGGTTCCTGTHk2-N313S-SENSE: (SEQ ID NO: 49)5′-CCCTGAGGACACGTACTACTCTGGCAACCCGTGGTTCCTGT Hk2-N313G-SENSE:(SEQ ID NO: 50) 5′-CCCTGAGGACACGTACTACGGTGGCAACCCGTGGTTCCTGTHk2-N315Q-ANTISENSE: (SEQ ID NO: 51)5′-AGGTGCACAGGAACCACGGTTGGCCGTTGTAGTACGTGTCC Hk2-N315E-ANTISENSE:(SEQ ID NO: 52) 5′-AGGTGCACAGGAACCACGGTTCGCCGTTGTAGTACGTGTCCHk2-N315R-ANTISENSE: (SEQ ID NO: 53)5′-AGGTGCACAGGAACCACGGTCTGCCGTTGTAGTACGTGTCC Hk2-F318Y-ANTISENSE:(SEQ ID NO: 54) 5′-CGGCAGCCAAGGTGCACAGATACCACGGGTTGCCGTTGTAGHk2-Q409P-SENSE: (SEQ ID NO: 55)5′-CGACAAGTCTGATGGCGAGCCACTTTCCGCTCGCGACCTGA Hklib3: Hk3-D336X-SENSE:(SEQ ID NO: 56) 5′-CGATGCTCTATACCAGTGGNNKAAGCAGGGGTCGTTGGAGGHk3-K337X-SENSE: (SEQ ID NO: 57)5′-TGCTCTATACCAGTGGGACNNKCAGGGGTCGTTGGAGGTCA Hk3-Q338X-ANTISENSE:(SEQ ID NO: 58) 5′-CTGTGACCTCCAACGACCCGNNCTTGTCCCACTGGTATAGAHk3-G339X-SENSE: (SEQ ID NO: 59)5′-ATACCAGTGGGACAAGCAGNCUTCGTTGGAGGTCACAGATG Hk3-S340X′-ANTISENSE:(SEQ ID NO: 60) 5′-ACACATCTGTGACCTCCAAANTCCCCTGCTTGTCCCACTGGHk3-S340X″-ANTISENSE: (SEQ ID NO: 61)5′-ACACATCTGTGACCTCCAAANCCCCCTGCTTGTCCCACTGG Hk3-L341X-SENSE:(SEQ ID NO: 62) 5′-GTGGGACAAGCAGGGGTCGNUUGAGGTCACAGATGTGTCGCHk3-K352Q-SENSE: (SEQ ID NO: 63)5′-TGTGTCGCTGGACTTCTTCCAAGCACTGTACAGCGATGCTG Hk3-K352R-SENSE:(SEQ ID NO: 64) 5′-TGTGTCGCTGGACTTCTTCAGAGCACTGTACAGCGATGCTGHk3-A353D-ANTISENSE: (SEQ ID NO: 65)5′-TAGCAGCATCGCTGTACAGATCCTTGAAGAAGTCCAGCGAC Hk3-A353S-ANTISENSE:(SEQ ID NO: 66) 5′-TAGCAGCATCGCTGTACAGAGACTTGAAGAAGTCCAGCGACHk3-S356P-SENSE: (SEQ ID NO: 67)5′-ACTTCTTCAAGGCACTGTACCCAGATGCTGCTACTGGCACCT Hk3-S356N-SENSE:(SEQ ID NO: 68) 5′-ACTTCTTCAAGGCACTGTACAAUGATGCTGCTACTGGCACCTAHk3-S356D-SENSE: (SEQ ID NO: 69)5′-ACTTCTTCAAGGCACTGTACGAUGATGCTGCTACTGGCACCTA Hk3-D357S-ANTISENSE:(SEQ ID NO: 70) 5′-GAGTAGGTGCCAGTAGCAGCAGAGCTGTACAGTGCCTTGAAGAHk3-A359S-SENSE: (SEQ ID NO: 71)5′-GGCACTGTACAGCGATGCTTCTACTGGCACCTACTCTTCGT Hk3-T360V-ANTISENSE:(SEQ ID NO: 72) 5′-TGGACGAAGAGTAGGTGCCAACAGCAGCATCGCTGTACAGTHk3-G361X-SENSE: (SEQ ID NO: 73)5′-TGTACAGCGATGCTGCTACTNCTACCTACTCTTCGTCCAGTTC Hk3-T362R-ANTISENSE:(SEQ ID NO: 74) 5′-GTCGAACTGGACGAAGAGTATCTGCCAGTAGCAGCATCGCTGHk3-S364X-SENSE: (SEQ ID NO: 75)5′-TGCTGCTACTGGCACCTACNNKTCGTCCAGTTCGACTTATAG Hk3-S365X-SENSE:(SEQ ID NO: 76) 5′-TGCTACTGGCACCTACTCTNNKTCCAGTTCGACTTATAGTAGHk3-S366T-ANTISENSE: (SEQ ID NO: 77)5′-ATGCTACTATAAGTCGAACTAGTCGAAGAGTAGGTGCCAGTA Hk3-S368X-ANTISENSE:(SEQ ID NO: 78) 5′-TCTACAATGCTACTATAAGTAGNACTGGACGAAGAGTAGGTGHk3-T369X-SENSE: (SEQ ID NO: 79)5′-CTACTCTTCGTCCAGTTCGNNKTATAGTAGCATTGTAGATGCC Hk3-S371X-ANTISENSE:(SEQ ID NO: 80) 5′-TTCACGGCATCTACAATGCTATNATAAGTCGAACTGGACGAAGHk3-S372X-SENSE: (SEQ ID NO: 81)5′-CGTCCAGTTCGACTTATAGTNNTATTGTAGATGCCGTGAAGAC

To the mix of Dnase treated DNA and oligos was added nucleotides, PCRbuffer and Taq/Pwo polymerase. A PCR assembly reaction was performed,using first 94° C. for 2 min., then 35-40 cycles with the followingincubation times: 94° C., 30 sec.; 45° C., 30 sec.; 72° C., 60 sec; thenfinally 72° C. for 5 min.

An PCR amplification reaction was performed with 1 uL of the assemblyreaction as template, and adding primers that anneal to the regionsflanking the AMG gene. Parameters: first 94° C. for 2 min., then 35-40cycles with the following incubation times: 94° C., 30 sec.; 55° C., 30sec.; 72° C., 90 sec; then finally 72° C. for 10 min.

The resulting PCR product was purified from a 1% agarose gel, mixed withlinearized vector and transformed into competent yeast cells, asdescribed above.

Example 4 Specific Activity

AMG G2 variants were constructed as described above in Example 1. Thespecific activity as k_(cat) were measured on purified samples at pH4.3, 37° C., using maltose and maltohepatose as substrate as describedin the “Materials & Methods” section above. The specific activity asAGU/mg were also measured at pH 4.3, 37° C., using maltose as substrateas described in the “Materials & Methods” section above.

Kcat (sec.−1) Variant Maltose Maltoheptaose AMG G2 (wt) 6.0 38 N110T 9.727.8 V111P 12.0 43.2 S119P 6.2 44.0 G127A 21.0 40.0 G207N 30.5 36.3Variant AGU/mg AMG G2 (wild type) 1.8 N110T 3.5 V111P 3.1 S119P 2.1G127A 5.8 G207N 5.7 L3N 2.3 S56A 2.6 A102* 2.5 D403S 2.2 I18V + T51S +S56A + V59T + L60A 3.3 S119P + Y402F 2.7 S119P + I189T + Y223F + F227Y +Y402F 3.0

Example 5 Thermostability at 70° C.

An AMG G2 S119P variant was constructed using the approach described inExample 1.

The thermostability was determined as T_(1/2) using Method I, and as %residual activity after incubation for 30 minutes in 50 mM NaOAc, pH4.5, 70° C., 0.2 AGU/ml, as described in the “Material & Methods”section above. The result of the tests are listed in the Table below andcompared to the wild-type A. niger AMG G2.

Residual T_(1/2) A. niger AMG (Enzyme) activity (%) (min.) S119P variant22 17 wild-type (SEQ ID NO: 2) 13 8

Example 6 Thermostability at 68° C.

AMG G2 variants were constructed using the approach described in Example3, except for variants nos. 1 and 2 in the Table below, which wereprepared by shuffling as described in WO 95/22625 (from AffymaxTechnologies N.V.).

The thermostability was determined as T½ using method I at 68° C. asdescribed in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Evaluation ofvariants was performed on culture broth after filtration of thesupernatants.

T½ A. niger AMG G2 T½ (wild type) Variant (min) (min) 1 A246T + T72I11.3 8.5 2 G447S + S119P 11.4 7.9 3 E408R + A425T + S465P + T494A 8.68.1 4 E408R + S386N 12.6 8.9 5 T2P 9.3 8.5 6 T2Q + A11P + S394R 10.7 8.57 T2H 9.5 8.9 8 A11E + E408R 12.7 9.3 9 T2M + N9A + T390R + D406N +L410R 10.7 8.5 10 A393R 17.7 8.4 11 T2R + S386R + A393R 14.1 8.4 12A393R + L410R 14.7 7.9 13 A1V + L66R + Y402F + N427S + S486G 11.7 8.5 14T2K + S30P + N427M + S444G + V470M 11.4 8.4

Thermostability at 70° C. on Purified Samples.

Enzyme T½ (min) 15 AMG G2 (wild type) 7.4 16 T2E + T379A + S386K + A393R11.6 17 E408R + S386N 10.2 18 T2Q + A11P + S394R 9.8 19 A1V + L66R +Y402F + N427S + S486G 14.1 20 A393R 14.6 21 T2R + S386R + A393R 14.1 22A393R + L410R 12.9 23 Y402F 10.1

Example 7 Thermostability at 68° C.

AMG G2 variants were constructed by shuffling using the approachdescribed in Example 3 followed by shuffling of positive variants.

The thermostability was determined as T½ using method I at 68° C. asdescribed in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Evaluation ofvariants was performed on culture broth after filtration of thesupernatants.

T½ A. niger AMG G2 T½ (wild type) Variant (min) (min) 24 PLASD^(i) +V59A + A393R + T490A 27.2 6.8 i = N-terminal extension

Example 8 Thermostability at 68° C.

AMG G2 variants were constructed using the approach described in Example3. The thermostability was determined as T½ using method I at 68° C. asdescribed in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Evaluation ofvariants was performed on culture broth after filtration of thesupernatants.

T½ A. niger AMG G2 T½ wild-type Variant (min) (min) 25 D357S + T360V +S371H 6.6 5.9 26 N313G + F318Y 8.9 5.9 27 S356P + S366T 7.3 5.8 28S340G + D357S + T360V + S386P 7.2 5.8

Example 9 Thermostability at 70° C.

An AMG G2 variants was constructed using the approach described inExample 1 and evaluated as semi-purified (filtration of culture brothfollowed by desalting on a G-25 column) samples.

The thermostability was determined as % residual activity using Method Iin 50 mM NaOAc, pH 4.5, 70° C., as described in the “Material & Methods”section above. The result of the test is listed in the Table below andcompared to the wild-type A. niger AMG G2.

Enzyme T½ (min) 29 AMG G2 (wild type) 7 30 Y402F + S411V 60 31 S119P +Y402F + S411V 115 32 S119P + Y312Q + Y402F + T416H 50

Example 10 Thermostability at 70° C. in Presence of 30% Glucose

AMG G2 variants were constructed using the approach described in Example3. The thermostability was determined as T½ using method II at 70° C. asdescribed in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions.

Enzyme T½ (hr) 33 AMG G2 (wild type) 1.5 34 Y402F 2.5 35 A393R 4.0 36T2R + S386R + A393R 2.0 37 PLASD (N-terminal) + V59A + A393R + T490A16.0

Example 11 Saccharification Performance of AMG VariantsS119P+Y402F+S411V and PLASD(N-Terminal)+V59A+A393R+T490A, Respectively

Saccharification performance of the AMG variants S119P+Y402F+S411V andPLASD(N-terminal)+V59A+A393R+T490A, respectively, both having improvedthermostability are tested at 70° C. as described below.

Reference enzyme is the wild-type A. niger AMG G2. Saccharification isrun under the following conditions:

Substrate 10 DE Maltodextrin, approx. 30% DS (w/w) Temperature 70° C.Initial pH 4.3 (at 70° C.) Enzyme dosage 0.24 AGU/g DSSaccharification

The substrate for saccharification is made by dissolving maltodextrin(prepared from common corn) in boiling Milli-Q water and adjusting thedry substance to approximately 30% (w/w). pH is adjusted to 4.3.Aliquots of substrate corresponding to 15 g dry solids are transferredto 50 ml blue cap glass flasks and placed in a water bath with stirring.Enzymes are added and pH re-adjusted if necessary. The experiment is runin duplicate. Samples are taken periodically and analysed at HPLC fordetermination of the carbohydrate composition.

1. A non-naturally occurring glucoamylase comprising a substitution atone or more position(s) selected from the group consisting of: 388, 390,394, 397, 398, 402, 403, 404, 405, 406, 409, 412, 413, and 414, wherein(a) the glucoamylase has at least 90% sequence identity to SEQ ID NO: 2;(b) the glucoamylase has glucoamylase activity; and (c) the sequence ofSEQ ID NO: 2 is used for position numbering.
 2. The glucoamylase ofclaim 1, which has at least 95% sequence identity to SEQ ID NO:
 2. 3.The glucoamylase of claim 1, which has at least 97% sequence identity toSEQ ID NO:
 2. 4. The glucoamylase of claim 1, which comprises a set ofmutations selected from the group consisting of:A1V+L66R+Y402F+N427S+S486G, T2M+N9A+T390R+D406N+L410R, T2Q-A11P+S394R,T2R+L66V+S394P+Y402F, S119P+I189T+Y223F+F227Y+Y402F,S119P+Y312Q+A393R+Y402F+S411V+T416H, S119P+Y312Q+Y402F+S411V+T416H,S119P+Y312Q+Y402F+T416H, S119P+A393R+Y402F+S411V,S119P+A393R+Y402F+S411V+T416H, S119P+Y402F, S119P+Y402F+S411V, andY402F+S411V.
 5. The glucoamylase of claim 1, which is a variant ofAspergillus niger G1 glucoamylase.
 6. The glucoamylase of claim 1, whichis a variant of a truncated glucoamylase.
 7. A process for convertingstarch or partially hydrolyzed starch into a syrup containing dextrose,comprising saccharifying a starch hydrolyzate in the presence of aglucoamylase of claim
 1. 8. A process of saccharifying a liquefiedstarch solution, comprising saccharifying the liquefied starch solutionwith a glucoamylase of claim
 1. 9. A non-naturally occurringglucoamylase comprising a substitution at one or more position(s)selected from the group consisting of: 389, 391, 392, 393, 395, 396,400, 401, 407, 408, 410, and 411, wherein (a) the glucoamylase has atleast 90% sequence identity to SEQ ID NO: 2; (b) the glucoamylase hasglucoamylase activity; and (c) the sequence of SEQ ID NO: 2 is used forposition numbering, wherein the substitution is not E389D/Q, H391W,A392D, A393P, N395Q, G396S, E400Q/C, Q401E, G407D, E408P, L410F,S411A/G/C/H/D.
 10. The glucoamylase of claim 9, which has at least 95%sequence identity to SEQ ID NO:
 2. 11. The glucoamylase of claim 9,which has at least 97% sequence identity to SEQ ID NO:
 2. 12. Theglucoamylase of claim 9, which comprises a set of mutations selectedfrom the group consisting of: T2E+T379A+S386K+A393R, T2R+S386R+A393R,A11E+E408R, V59A+A393R+T490A+PLASD (N-terminal extension), S119P+A393R,S386N+E408R, A393R+L410R, A393R+S411V, and E408R+A425T+S465P+T494A. 13.The glucoamylase of claim 9, which is a variant of Aspergillus niger G1glucoamylase.
 14. The glucoamylase of claim 9, which is a variant of atruncated glucoamylase.
 15. A process for converting starch or partiallyhydrolyzed starch into a syrup containing dextrose, comprisingsaccharifying a starch hydrolyzate in the presence of a glucoamylase ofclaim
 9. 16. A process of saccharifying a liquefied starch solution,comprising saccharifying the liquefied starch solution with aglucoamylase of claim
 9. 17. A non-naturally occurring glucoamylase,comprising a substitution at position 399 with A, R, N, D, C, Q, E, G,H, I, L, K, F, M, P, T, W, Y, or V, wherein (a) the glucoamylase has atleast 90% sequence identity to SEQ ID NO: 2; (b) the glucoamylase hasglucoamylase activity; and (c) the sequence of SEQ ID NO: 2 is used forposition numbering.
 18. The glucoamylase of claim 17, which is a variantof Aspergillus niger G1 glucoamylase.
 19. The glucoamylase of claim 17,which is a variant of a truncated glucoamylase.
 20. A process forconverting starch or partially hydrolyzed starch into a syrup containingdextrose, comprising saccharifying a starch hydrolyzate in the presenceof a glucoamylase of claim
 17. 21. A process of saccharifying aliquefied starch solution, comprising saccharifying the liquefied starchsolution with a glucoamylase of claim 17.